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Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli
Accepted Manuscript Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli Qiusheng Zhang, Binglian Xu, Jiajia Pan, Danyang Liu, Ruoxian Lv, Dongchun Yan PII:
S1046-5928(18)30078-0
DOI:
https://doi.org/10.1016/j.pep.2019.02.007
Reference:
YPREP 5388
To appear in:
Protein Expression and Purification
Received Date: 9 February 2018 Revised Date:
16 December 2018
Accepted Date: 6 February 2019
Please cite this article as: Q. Zhang, B. Xu, J. Pan, D. Liu, R. Lv, D. Yan, Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli, Protein Expression and Purification (2019), doi: https://doi.org/10.1016/j.pep.2019.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression and active testing of VP7 from GCRV (Grass carp reovirus)
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fused with cholera toxin B Subunit in Rice Calli Qiusheng Zhang *, Binglian Xu, Jiajia Pan, Danyang Liu, Ruoxian Lv, Dongchun Yan
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Runing title: Expression and active testing of CTB-VP7 in rice calli
Ludong University. Yantai, Shandong Province, 264025, China Corresponding Author: Qiusheng Zhang E-mail:
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*
[email protected]
Phone: 086-27-68780736
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Abstract
Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from
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GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based
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oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear
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genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54%±0.43 of the total
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soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different
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temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37℃, and approximately half of the binding affinity remained at 25℃. Our results
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suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.
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Keywords
Grass Carp Reovirus (GCRV), VP7, Cholera toxin B subunit(CTB), rice calli,
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Plant-based vaccine
1. Introduction
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Grass carp, Ctenopharyngodon idellus (Valenciennes), is an economically important
species cultivated in China. However, grass carp are vulnerable to GCRV infection, resulting in a greater than 80% mortality rate and significant economic losses [1,2,3]. GCRV prevention and control are essential to the continued development of aquaculture, both economically and environmentally [4]. Ultimately, effective vaccines will probably
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become an important way to resolve GCRV in the successful development of the intensive grass carp farming industry. Grass carp have cells that are phenotypically and functionally similar to gut
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intraepithelial and systemic T cells [5]. Currently, the oral vaccination route has the advantage that the vaccine is inexpensive and easy to administer without causing any stress to the fish [6,7]. Some results support that transgenic plants could serve as a
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promising candidate for the future development of vaccines. Therefore, there is a need to exploit plant biotechnology for cost-effective oral fish vaccine development in plants,
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particularly in edible crops. The application of plant biotechnological tools for fish vaccine development is important for aquaculture because fish vaccines must be produced at a low cost and easily scalable, making them accessible and affordable for not only the aquaculture industry worldwide but also for improving the conditions for small
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fish farmers in developing countries [8,9]. Therefore, oral vaccination with antigens included in the feed would be the ideal method of vaccine delivery to fish. The mature GCRV particle is composed of 7 proteins, VP1-VP7. Among the 7
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structural proteins, there are 5 protein components (VP1, VP2, VP3, VP4 and VP6) involved in forming the viral core, and two proteins, VP5 and VP7, involved in the outer
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capsid shell [10]. Based on a comparative study between GCRV and other strains, VP7 presented high similarity [2].VP7 was expressed in Escherichia coli, suggesting that VP7 could be used as a potential subunit vaccine against GCRV infection [1]. The VP7 protein was reported to be involved in viral infection and could be suitable for developing a subunit vaccine for the control of GCRV infection [11]. Furthermore, the homology of the VP7 gene among different GCRV strains was 99%, and the conservation was better
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than that of VP5 and VP6, so the VP7 vaccine could resist the attack of the GCRV strain to a certain degree [12]. Therefore, it is appropriate to select VP7 as the target vaccine for GCRV.
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Cholera toxin B subunit (CTB) is known to be a potent mucosal adjuvant; it is arranged in a pentameric ring and contains five receptor-binding pockets for high-avidity association with cellular membranes containing GM1 [13]. CTB has been used as a
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carrier and an adjuvant for fused antigen proteins in plant-based vaccines to stimulate the uptake and immune response upon feed-mediated oral immunization [7]. It is expected
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that the plant-produced CTB-VP7 fusion proteins could be efficiently taken up by a mucosal immune system, resulting in an increased immune response against GCRV. So far, there are few reports describing fish vaccine antigens expressed in edible crops by plastid engineering, although the technology for plastid engineering of edible crops such
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as lettuce, tomato, potato, etc. has been developed and used to express a number of foreign proteins [9]. The use of plants for the expression and delivery of CTB-VP7 is attractive for creating oral vaccines against GCRV in grass fish.
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In this study, we investigated a recombinant protein in which one CTB and one VP7 were driven by the ubiquitin promoter. Then, the potential recombinant CTB-VP7 fusion
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protein was evaluated for use as a vaccine against GCRV infection [14]. Our results showed that rice calli could highly express the fusion protein of CTB-VP7 and have biological activity, suggesting that it could possibly be used as an orally administered vaccine against GCRV in grass fish.
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2. Materials and methods 2.1. Synthesis and production of the VP7 recombinant protein The sequence coding for the VP7 protein from GCRV was synthesized according to
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the deposited sequences in GenBank (GenBank AF403396). The VP7 gene was inserted into BamH I sites in pET28a (Novagen, Darmstadt, Germany) to construct pET28a-VP7. For expression, the plasmids pET28a and pET28a-VP7 were transformed into BL21
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(DE3) cells and induced with 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). For purification, the ProBondTM Purification System (Invitrogen, Waltham, MA, USA)
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was used according to the manufacturer’s instructions. The recombinant protein was finally eluted from the column with the same buffer containing 150 mM imidazole. The VP7 fusion protein (rVP7) concentration was determined by the Bradford assay [15]. Whole BL21 cell lysates before and after induction, supernatant and precipitate after
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sonicating, rVP7 were added equal volume of 2×SDS loading buffer, and boiled for 10.0 min in a boiling water bath, followed by separation on 12% SDS-PAGE and visualization
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by staining with Coomassie brilliant blue R-250.
2.2. Preparation of the polyclonal antibody and Western blot
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Before immunization, 0.5-1.0 ml of blood from Balb/c mice was collected as a
negative serum control. A polyclonal antibody against rVP7 was generated as we described previously[16]. Whole BL21 cell lysates and rVP7 were separated on SDS-PAGE and proteins were subsequently transferred to nitrocellulose membrane (Thermo Scientific, Waltham, MA, USA) using a Mini Tran-Blot® Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA) at 80V for 60 min. After blocking for 2h in
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5% skimmed milk in phosphate-buffered saline (PBS), membrane was incubated overnight at 4°C with anti-rVP7 mouse polyclonal antibody (1: 5000). The secondary antibody HRP-conjugated goat anti-rabbit IgG (Sangon, Shanghai, China) were
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employed to react with the primary antibody for 1 h at room temperature. The protein bands were then visualized using a SuperSignal West Dura solution Extended Duration
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Substrate (Thermo Scientific, Waltham, MA, USA).
2.3. Construction and expression of the CTB-VP7 fusion gene
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For the CTB-VP7 fusion gene, the CTB fragment and VP7 gene were combined with a flexible connection linker. The synthetic CTB-VP7 was modified based on plant-optimized codon usage and fused to a translation signal (the Kozak sequence, GCCACC) at the front of the start codon and the ER retention signal,
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SEKDEL(TCTGAGAAAGATGAGCTA), at the 3’-terminus. For subsequent vector manipulations, BamH I restriction sites were introduced at the 5’- and 3′-termini of the CTB-VP7 fragment. The DNA products were then inserted into the bacterial expression
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vector pET28a, and the correct clone bearing the CTB-VP7 fusion gene was confirmed by gene sequencing. Then the plasmid pET28a-CTB-VP7 was transformed into BL21 (DE3)
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cells. Expression, purification and Western blot of the fusion protein (rCTB-VP7) were performed according to the above instructions. rCTB-VP7 was subsequently eluted from the column with the same buffer containing 200 mM imidazole.
2.4. Rice plant transformation and regeneration Agrobacterium-mediated genetic transformation experiments were carried out using
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the EHA105 strain harboring pUbi-CTB-VP7. The rice cultivar ‘ZH11’ was used for genetic transformation. This was performed under the control of the maize ubiquitin-1
generated and grown as we described previously [17].
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(Ubi) promoter and nopaline synthase (nos) terminator. Transgenic rice calli were
2.5. Genomic PCR analysis of the insertion of the CTB-VP7 fusion gene in the plant
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genome
DNA insertion was confirmed by genomic PCR analysis. Rice genomic DNA was
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extracted from calli, and subsequent PCR analysis was performed as we described previously [15]. PCR was performed using ExTaq polymerase (Takara, Dalian, China) and two primers, VP7F and VP7R (VP7F; 5′-GCAGAACATCACTGACCTGTGTG-3′, VP7R; 5′- CATAGCTCATCTTTCTCAGAATC-3′). Regenerated nontransgenic rice
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plants were used as negative controls. Twenty-five cycles of PCR were performed, and the conditions were as follows: denaturation at 94°C for 1.0 min, annealing at 56°C for 1.0 min and extension at 72°C for 1.0 min. PCR products were detected by 1.5% agarose
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gel electrophoresis followed by ethidium bromide (EB) staining.
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2.6. RNA analysis
Total RNA was extracted from rice calli using the Plant RNA Purification Reagent
(Invitrogen, USA) according to the manufacturer’s instructions. Total RNA was incubated with RNase-free DNase I (Life Technology, Gaithersburg, USA) to eliminate contaminated genomic DNA before being reverse transcribed into cDNA using oligo d(T)18 primers and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). The
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expression of the CTB-VP7 fusion gene was determined using qRT-PCR. The 20 µl PCR reaction mixtures contained 1 µl of each primer (0.5 µM), 2 µl of template, 6 µl of H2O and 10 µl of 2×iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on the
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Bio-Rad CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA). The PCR conditions involved an initial denaturation cycle of 95°C for 3 min, followed by 25 cycles of 95°C for 10 s, 55°C for 30 s, and plate read. Each sample was run in triplicate.
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Relative gene expression levels were determined by the comparative threshold cycle method (2-∆∆ C t) with β-actin used as the reference gene. Two PCRs were performed with combination
of
primers
(F1⁄R1)
as
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a
follows:
F1
(5’-
GAAGTACCAGGTAGCCAGCA-3’) and R1 (5’-TTGTGTACAGGGTAAGGCGT-3’). The β-actin gene was amplified using an Actin-F⁄Actin-R primer set (5′-GTC AGC AAC TGG GAT GAT ATG G-3′/5’-TCT TCC TTG CTC ATC CTG TCA G-3’) as an internal
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control to normalize the amount of rice callus RNA.
2.7. Crude protein extraction from nontransgenic and transgenic rice calli
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Approximately 100 mg of rice callus tissue was frozen in liquid nitrogen, ground into a fine powder, and resuspended in 200 µl of protein extraction buffer [PBST plus 10
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µg/ml leupeptin and 1.0 mM phenylmethylsulfonyl fluoride]. Samples were centrifuged at 14,000 rpm in a microcentrifuge for 10 min at 4°C. Total soluble protein (TSP) concentrations were determined in supernatants by the Bradford assay [16].
2.8. SDS-PAGE and Western blot analysis of CTB-VP7 protein in rice calli
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The crude protein was extracted from rice calli as previously described [17]. To assess the integrity of CTB-VP7, Western blotting was carried out using TSP plant extracts from WT (wild-type), VT (vector-transgenic), and transgenic plants, and 50 ng
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of pure rCTB-VP7 was used as a control. Samples were boiled for 10.0 min and resolved by SDS-PAGE. At the same time, two repeated experiments were performed, one for
performed according to the above instructions.
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staining and one for Western blotting, using an anti-rVP7 antibody (1: 5000) were
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2.9. ELISA and GM1 binding assay of the CTB-VP7 protein in rice calli
ELISA was performed to quantify the amount of CTB-VP7 in rice calli. The CTB-VP7 content in the crude extract and the ability to bind GM1 were determined using
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a ganglioside-dependent ELISA [18]. TSP extracts were coated on ELISA plates.
2.10. Purification of the CTB-VP7 protein from rice calli Equal amounts of rice callus tissue (20.0 g) from five independent lines was ground
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in liquid nitrogen and homogenized in 20 ml of protein extraction buffer. Purification of CTB-VP7 from the crude protein extraction of rice calli was carried out as described in
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Su et al. [19].
2.11. GM1- binding assay of the CTB-VP7 protein in bacteria and rice calli The concentrations of rCTB-VP7 and CTB-VP7 from prokaryotes and rice calli
were determined using the Bradford assay with a calibration curve base on BSA [20].
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GM1-ELISA was conducted to determine the affinity of rCTB-VP7 and CTB-VP7 for the GM1 receptor. The two proteins were quantified by ELISA with the rVP7 antibody to ensure that the amounts of the two proteins were consistent. The wells of
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plates were coated with 100 µl of GM1 (0.3 µg/ml) (Sigma-Aldrich, MO, USA). After washing, the wells were blocked with 200 µl of blocking buffer containing milk and then
the previously described methods for indirect ELISA.
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incubated for 2 h at 37°C. The remaining procedures were conducted in accordance with
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2.12. Assay of the biological activity of the CTB-VP7 fusion protein from rice calli Indirect ELISA was performed to check the biological activity of CTB-VP7 from rice calli at different temperatures. To test the relative binding affinity of CTB-VP7 with GM1 receptors, microtiter plates were coated with 100 µl of GM1 (0.3 µg/ml) and
3. Results
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incubated at 4℃, 25℃, 37℃, 42℃, 47℃, and 100℃ for 2 h individually.
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3.1. Detection of VP7 expressed in E. coli and its antibody E. coli strain BL21 transformed with pET28a-VP7 was induced with IPTG for 3 h. As
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expected, the molecular weight of the expressed protein was approximately 37.0 kDa, as determined by SDS-PAGE. After ultrasonic disruption, the specific protein could only be detected in inclusion bodies but not in the supernatant, suggesting that rVP7 is expressed mainly in inclusion bodies inside bacterial cells. rVP7 was purified through a Ni2+ affinity column and then used to prepare a mouse polyclonal antibody. The mouse antibody titer against rVP7 was higher than 1:100,000, as determined by ELISA. No cross-reaction was
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detected for the negative control, indicating that the rVP7 antibody was successfully prepared. Western blotting was carried out and showed that the positive signal for the
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polyclonal antibody recognizing the rVP7 was at the expected size of approximately 37.0 kDa. In contrast, the control contained no signal in the assay. The results suggested that the prepared antibody could be used to detect rVP7. Western blotting indicated that the
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expressed rVP7 was specifically recognized by the mouse anti-rVP7 polyclonal antibody
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(Fig. 1).
3.2. CTB-VP7 fusion gene expressed in E. coli
E. coli strain BL21 transformed with pET28a-CTB-VP7 was induced with IPTG for 3 h. After ultrasonic disruption, the specific protein could only be detected in inclusion
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bodies but not in the supernatant, suggesting that the rCTB-VP7 was expressed mainly in inclusion bodies inside bacterial cells. rCTB-VP7 was purified through a Ni2+ affinity column. A specific band of approximately 49.0 kDa representing the rCTB-VP7 could be
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detected by SDS-PAGE and Western blotting with a mouse anti-rVP7 antibody, indicating that rCTB-VP7 was correctly expressed in E. coli (Fig. 2). Western blotting
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indicated that the expressed rCTB-VP7 was specifically recognized by the mouse anti-rVP7 polyclonal antibody.
3.3. Transformation of rice calli with CTB-VP7 The Kozak sequence (GCCACC) in front of the start codon and an ER retention signal (SEKDEL) before the stop codon were applied to the CTB-VP7 fusion gene to
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improve the expression level of the recombinant protein in transgenic plants. The binary recombinant plasmid pUbi-CTB-VP7, which was designed to express CTB-VP7 under the control of a ubiquitin promoter, was constructed (Fig. 3a). These plasmids
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(pUbi-CTB-VP7, pUbi) were introduced into rice plants by Agrobacterium. tumefaciens-mediated gene transformation respectively. After infection with A. 34
antibiotic-resistant
rice
scutellum-derived/embryogenic calli in 40 days.
calli
proliferated
from
150
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tumefaciens,
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3.4. Detection of the CTB-VP7 gene in transgenic rice calli
Characterization of transgenic callus integration of CTB-VP7 into rice genomes was confirmed by genomic PCR, which showed amplification of a 1.2 kb fragment corresponding to the CTB-VP7 gene amplified from the vector. Of the 34 regenerated rice
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calli tested, 15 lines were positive for amplification (Fig. 3b). No amplification was obtained using non-transgenic rice lines.
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3.5. Detection of CTB-VP7 transcripts in transgenic rice calli To analyze CTB-VP7 transgene transcript levels, qRT-PCR was performed using two
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pairs of primers and RNA from rice calli. Primers were designed in such a way that one pair could detect the transcript levels in addition to whole CTB-VP7, facilitating the evaluation of two coordinating gene expression levels. Transcript levels were similar among transgenic lines with the exception of CTB-VP7 line 11, which has an expression level 1.25- to 2.91-fold higher than other lines. Thus, coordinated expression of the
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CTB-VP7 sequences was equally effective at the transcript level. The transcript levels in
3.6. Detection of the CTB-VP7 protein in transgenic rice calli
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CTB-VP7 line 11 was 2.91 that of line 26 (Fig. 4).
The expression level of the CTB-VP7 protein in transformed calli was determined via ELISA in accordance with the methods established previously [17]. The analysis showed
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that CTB-VP7 protein was expressed in the rice calli at levels ranging from 0.56 to 2.17%, an average of 1.54% of the total soluble protein. Western blot analysis showed
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that the heterologous protein contributed by CTB-VP7 had an apparent molecular weight of 45 kDa, which is slightly larger than the theoretical counterpart (43 kDa), suggesting that the recombinant protein had been processed in plant cells (Fig. 5). No corresponding band could be visualized in WT and VT plants. In addition, the recombinant protein was
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recognized specifically by the VP7 polyclonal antibody; thus, the band was recognized as CTB-VP7 expressed in rice calli.
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3.7. Binding assay of the plant-produced CTB-VP7 protein to GM1 GM1-ELISA was conducted to determine the affinity of CTB-VP7 for the GM1
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receptor. The crude protein extract with CTB-VP7 produced in rice calli demonstrated a strong affinity for GM1 but not for BSA. The crude protein extracts from WT, VT and PBS rice calli showed no or very weak affinity for GM1. Thus, the strong relative binding efficiency of CTB-VP7 in crude protein extract for GM1 indicated that the transgenic rice callus-derived CTB subunit interacted with GM1 (Fig. 6).
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3.8. Binding affinity of the CTB-VP7 fusion protein from rice calli and bacteria The function of the CTB-VP7 fusion protein was correlated with the ability to bind to GM1. The binding affinity of rCTB-VP7 and CTB-VP7 to GM1 was determined by
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ELISA. Within the linear range of binding at variable molar concentrations of GM1, the order of binding affinity was CTB-VP7> rCTB-VP7. Thus, CTB-VP7 showed a higher
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affinity towards GM1 than rCTB-VP7 (Fig. 7).
3.9. Temperature and binding affinity of the CTB-VP7 fusion protein
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The binding affinity of CTB-VP7 was determined by GM1-ELISA carried out at different temperatures, such as 4℃, 25℃, 37℃, 42℃, 47℃, and 100℃. Within the linear range of binding at the same concentrations of GM1, the order of binding affinity was 37℃>42℃>47℃>25℃>4℃>100℃. Thus, CTB-VP7 showed a higher affinity for GM1 at
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37℃ than at other temperatures. Maximum binding of CTB-VP7 to GM1 was reported to
(Fig. 8).
4. Discussion
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occur within 2 h at 37℃, and approximately half of the binding affinity remains at 25℃
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Oral vaccines have received increasing attention owing to their convenience and
effectiveness. In the case of developing a virus vaccine for grass fish, a plant-based vaccine is a good choice [5]. Compared with bacteria, plants are more able to express viral proteins because of their glycosylation process. This is very important because glycan moieties are an important factor in the generation of neutralizing antibodies. The recombinant proteins produced in plants have higher immunogenicity when administered
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with the plant cell wall components [21]. Fish culture in paddy fields has a long history and ecological significance in China. Furthermore, rice is a model plant, and the technology of genetic transformation is mature and convenient. The use of rice calli as
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bioreactors is more feasible because it does not involve assessing the ecological safety of transgenic plants. Therefore, the use of rice callus cells to produce oral GCRV vaccines is a better choice.
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The present study aimed to identify the genetic engineering vaccine potential of the outer shell protein VP7. As the outer shell protein, VP7 might have key effects on viral
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infection [22]. The fact that anti-VP7 polyclonal antibodies efficiently blocked viral infection here supported that VP7 was involved in viral infection and may be a good candidate for the development of a vaccine. Meanwhile, CTB has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for
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chemically or genetically conjugated autoantigens for the induction of oral tolerance [23,24]. Conjugation with CTB may greatly facilitate antigen delivery and presentation to the gut-associated lymphoid tissues (GALT) due to its affinity for the cell surface
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receptor GM1 located on cells of the GALT, including the membranous cells and enterocytes, for increased uptake and immunologic recognition [25]. Therefore, the
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CTB-VP7 fusion protein could generate protective serum and mucosal antibodies, resulting in an increased protective immune response of grass carp against GCRV infection.
The genome of transgenic rice calli was analyzed for genetic stability by PCR
analysis. It was confirmed that the CTB-VP7 coding sequence was successfully integrated into the genome of rice. Moreover, the results of the qRT-PCR detection of CTB-VP7
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showed that it was expressed in transgenic rice. The expression of the CTB-VP7 protein in different transgenic plants showed wide variation. This is consistent with the transcript level determined by real-time PCR [26]. The variation in expression is likely due to the
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integration of the transgene at different positions in the genomes of independent transgenic lines [27]. These different expression levels of the CTB-VP7 fusion gene among the transgenic plants are due to the different incorporation sites of the target gene
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in the chromosomes of individual plants, known as the ‘position effect’[28]. Our results indicated that the transgenic rice calli was stable enough to maintain its genetic integrity.
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The integrity of the CTB-VP7 protein was confirmed by Western blot analysis using an anti-rVP7 antibody. This further confirmed that the rice-callus-derived fusion protein retained its antigenic properties and displayed the antigenic determinants of both CTB and VP7. We successfully expressed CTB-VP7 in rice calli using an A.
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tumefaciens-mediated transformation method. In addition, the biological activity of the plant-produced CTB-VP7 for GM1 was confirmed by GM1-ELISA. However, the molecular mass of CTB-VP7 expressed in rice calli was approximately 45 kDa, which is
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smaller than the 6His-CTB-VP7 fusion protein (49 kDa) and slightly larger than its theoretical counterpart (43 kDa), suggesting that the recombinant protein had been
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processed in rice calli through processes such as glycosylation and phosphorylation [25]. This is in line with the major advantages of plant cells for their ability to provide eukaryotic posttranslational modifications and expression products that possess high bioactivity and immunogenicity. The crude extracts from the CTB-VP7 plant tissues were found to have biology activity based on an in vitro assay using GM1[29]. The nuclear-encoded CTB-VP7
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protein retains the pentameric structure of CTB as it binds to GM1 and is recognized by rVP7-specific antibodies. This is the first study describing the production of biologically active fish CTB-VP7 using transgenic plants. In light of the apparent aggregation of the
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two proteins, it may be useful to increase the size of the intermolecular linker (Gly4Ser)3 to further reduce steric hindrance to facilitate assembly of the fusion protein monomers into pentamers rather than oligomers of variable size [30]. The linker was designed to
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direct the protein folding [31]. In general, the antigenic activity of CTB-VP7 suggests that it was correctly assembled and functionally produced in the plant cell.
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Immunoblot analysis and GM1-ELISA experiments showed the probability that monomeric B subunits accumulate within the lumen of the ER of plant cells where self-assembly into oligomeric, possibly pentameric, GM1 bound forms occur [32]. Therefore, these results indicated that the extension of CTB plus a linker to VP7 did not
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prevent CTB pentamer formation and that both components of the fusion protein retained antigenicity. The binding assay showed that a pentameric CTB was formed with functionally correct folding and assembly of the chimeric CTB-VP7 expressed in rice
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calli [33]. The plant-synthesized CTB-VP7 shows a higher affinity for binding to GM1 compared to the bacterial rCTB-VP7. The glycosylation of the plant-expressed CTB-VP7
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was predicted to facilitate functionally more favorable folding of the CTB-VP7 in bacterial cells [34]. The chaperones, other folding enzymes and glycosylation in plant cells may lead to an increase in the affinity of CTB-VP7 for GM1 receptors compared to the nonglycosylated bacterial rCTB-VP7. The glycosylation of the plant-expressed CTB was predicted to facilitate functionally more favorable folding of the CTB in rice cell [35,36]. The plant-expressed
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CTB-VP7 bonded to GM1 with different affinities at different temperatures. Maximum binding of the cholera toxin to GM1 was found to occur at 37℃. These results indicated that temperature affects the spatial structure of the plant-expressed CTB-VP7 fusion
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protein, especially the configuration of CTB, and thus affects the binding power of the protein to GM1. Fishes live in water, and therefore, the temperature of the water has a strong influence on the physiological function of fish [37]. The suitable temperature
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range for fish growth is 15-30℃[38]. Within this temperature range, the binding of CTB-VP7 from rice calli to GM1 still has activity, but its activity is lower than that at
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37℃. Therefore, CTB-VP7 can be used as an oral vaccine for fish.
5. Conclusions
This study demonstrated that a GCRV VP7 capsid antigen coupled with CTB could
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be synthesized and assembled into a functional form in rice cells as a step towards developing an oral plant-based vaccine for grass fish to prevent GCRV. The strong GM1 binding affinity of the CTB-VP7 fusion protein suggests its potential as an oral vaccine,
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and future studies will address its immunogenic potential in grass fish. Our results provide a reliable system for further in vitro expression and assembly of GCRV particles
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using a plant expression system.
Conflicts of interest
The authors declare that they have no competing interests.
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Acknowledgments This work was supported by the Shandong Provincial Natural Science Foundation, China (No. ZR2016CM10), Key R & D program of Shandong Province, China (No.
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2018GSF121035) and A Project of Shandong Province Higher Educational Science and
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Technology, China (No. J13LE09)
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Figure Legends
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Fig1. VP7 fusion protein prokaryotic expression vector construction, expression, purification and test a,VP7 prokaryotic expression vector construction; M, DNA marker; 1,VP7 prokaryotic expression vector digested with BamHI; b, VP7-fusion prokaryotic expression; M, protein marker; 1,the no-induced germ total proteins; 2, the induced germ total proteins; C, Purification of rVP7; M, protein marker; 1, Supernatant after sonicating and centrifuging; 2, precipitate after sonicating and centrifuging; 3, purified rVP7, d, Western blot of the antibody from
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rVP7.
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rVP7. M, protein marker; 1, no-induced germ total proteins, 2, the purified rVP7; Arrows in b, c and d indicate
Fig2. CTB-VP7 fusion protein prokaryotic expression vector construction, expression, purification and test
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ACCEPTED MANUSCRIPT a, Schematic structure of the fusion gene CTB-VP7.CTB-VP7construct was engineered with a flexible peptide
linker between CTB and VP7; b, CTB-VP7 prokaryotic expression vector construction. M, DNA marker. 1, The
integration fusion gene fragment with the VP7 and CTB. 2,CTB-VP7prokaryotic expression vector digested with
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BamHI; c, CTB-VP7 fusion prokaryotic expression. M, Protein marker. 1, no-induced germ total proteins. 2,
induced germ total proteins. 3, Supernatant after sonicating and centrifuging. 4, Precipitate after sonicating and
centrifuging; d, SDS-PAGE of rVP7 and rCTB-VP7. M, protein marker. 1, rVP7. 2, rCTB-VP7; e, Western blot of
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rVP7 and rCTB-VP7 by rVP7 antibody. M, protein marker; 1, rVP7; 2, rCTB-VP7. Arrows in c ,d and e indicate
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rCTB-VP7.
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Fig 3. a: Schematic representation of designed constructs for expression of the CTB-VP7 gene in rice calli.
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HYG, Hygromycin B, was used for screening the transformants; the CTB-VP7 gene was driven by the Ubi promoter and terminated by the Nos terminator. LB and RB represent left and right borders respectively. b: Genomic DNA PCR analysis used to detect the CTB-VP7 gene in transgenic rice callus lines. M, DL2000 molecular markers; C, PCR control; VT, transformed rice calli with empty-vector; WT, nontransgenic rice calli; 4, 9, 11, 12, 15, 17, 26, different transgenic rice callus lines.
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Fig 4. The expression levels of CTB-VP7 by quantitative real-time PCR
The accumulation of CTB-VP7 transcripts was determined by qRT-PCR. Total RNA was isolated from
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rice calli of WT (wild type), VT (transformed rice calli with empty-vector) and transgenic rice callus lines (4, 11, 15, 17, 26). The constitutively expressed β-actin mRNA, set to 100%, was used as an internal standard to normalize relative amounts of transcripts in all experiments. The mRNA samples from five independently harvested rice callus lines were analyzed. Data showed the means±SE of five
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independent experiments.
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ACCEPTED MANUSCRIPT Fig 5. Total soluble protein extracts were resolved by SDS-PAGE under denaturing condition and subjected
to blotting and immunodection by anti-rVP7 serum. a: SDS-PAGE of crude protein extracted from the rice calli of empty vector, transgenic vector. b: Western blot examination of CTB-VP7 in different rice callus lines.
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26, different rice callus lines. Arrows in a and b indicate CTB-VP7.
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M, protein marker. C0, CTB-VP7 fusion protein; VT, transformed rice calli with empty-vector; 4, 11, 15, 17 and
Fig 6. GM1 binding CTB-VP7 from rice calli by ELISA
ELISA assay was performed by coating the wells of the microtiter plate with GM1 or BSA. Anti-rVP7
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antibody was added to identify the biological binding activity of plant-derived CTB-VP7 to GM1.
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WT, wild type rice calli; VT, transformed rice calli with empty-vector; 4, 11, 15, 17 and 26, transformed rice calli with CTB-VP7. Data are expressed as mean±SE. Error bars represent standard error of the mean.
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Fig 7. GM1 binding test of CTB-VP7 fusion protein from bacterial and rice calli
The purification of CTB-VP7 fusion protein from bacterial and rice calli, BSA as control, were serially diluted
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from 1 (1:20), 2 (1:50), 3 (1:100), 4 (1:200) to 5 (1:400) and incubated with anti-rVP7 antibody.
Fig 8. GM1 binding test of the CTB-VP7 fusion protein from rice calli under different temperatures
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The purification of CTB-VP7 fusion protein from rice calli, BSA as control, were serially diluted from 1 (1:20), 2 (1:50), 3 (1:100), 4 (1:200) to 5 (1:400) and incubated with anti-rVP7 antibody.
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S1046-5928(18)30078-0
DOI:
https://doi.org/10.1016/j.pep.2019.02.007
Reference:
YPREP 5388
To appear in:
Protein Expression and Purification
Received Date: 9 February 2018 Revised Date:
16 December 2018
Accepted Date: 6 February 2019
Please cite this article as: Q. Zhang, B. Xu, J. Pan, D. Liu, R. Lv, D. Yan, Expression and active testing of VP7 from GCRV (Grass carp reovirus) fused with cholera toxin B subunit in rice calli, Protein Expression and Purification (2019), doi: https://doi.org/10.1016/j.pep.2019.02.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expression and active testing of VP7 from GCRV (Grass carp reovirus)
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fused with cholera toxin B Subunit in Rice Calli Qiusheng Zhang *, Binglian Xu, Jiajia Pan, Danyang Liu, Ruoxian Lv, Dongchun Yan
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Runing title: Expression and active testing of CTB-VP7 in rice calli
Ludong University. Yantai, Shandong Province, 264025, China Corresponding Author: Qiusheng Zhang E-mail:
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*
[email protected]
Phone: 086-27-68780736
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Abstract
Grass carp reovirus (GCRV) is one of the most serious pathogens threatening grass carp (Ctenopharyngodon idellus) production and results in high mortality in China. VP7 from
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GCRV is involved in viral infection and could be suitable for developing vaccines for the control of GCRV infection. To obtain a genetically engineered vaccine and a plant-based
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oral vaccine and to evaluate their immune efficacy as an oral vaccine against GCRV, cholera toxin B subunit (CTB) of Vibrio cholerae fused to VP7 (CTB-VP7) was transformed into BL21(DE3) for expression. SDS-PAGE and Western blotting showed that the purified CTB-VP7 fusion protein (rCTB-VP7) was approximately 49.0 kDa. Meanwhile, CTB-VP7 was transformed into rice callus cells by Agrobacterium tumefaciens-mediated gene transformation. CTB-VP7 was integrated into the nuclear
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genome by PCR, and mRNA transcripts of CTB-VP7 were detected. ELISA and Western blot analyses revealed that the CTB-VP7 fusion protein (CTB-VP7) could be expressed in rice callus lines. The level of expression was determined to be 1.54%±0.43 of the total
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soluble protein. CTB-VP7 showed a binding affinity for monosialoganglioside(GM1), a receptor for CTB. CTB-VP7 showed a higher affinity towards GM1 compared to rCTB-VP7. CTB-VP7 bonded to GM1 with different affinities under different
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temperatures. Maximum binding of CTB-VP7 to GM1 was reported to occur within 2 h at 37℃, and approximately half of the binding affinity remained at 25℃. Our results
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suggest that CTB-VP7 could be produced in rice calli, increasing the possibility that edible plants can be employed in mucosal vaccines for protection against GCRV in aquaculture.
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Keywords
Grass Carp Reovirus (GCRV), VP7, Cholera toxin B subunit(CTB), rice calli,
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Plant-based vaccine
1. Introduction
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Grass carp, Ctenopharyngodon idellus (Valenciennes), is an economically important
species cultivated in China. However, grass carp are vulnerable to GCRV infection, resulting in a greater than 80% mortality rate and significant economic losses [1,2,3]. GCRV prevention and control are essential to the continued development of aquaculture, both economically and environmentally [4]. Ultimately, effective vaccines will probably
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become an important way to resolve GCRV in the successful development of the intensive grass carp farming industry. Grass carp have cells that are phenotypically and functionally similar to gut
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intraepithelial and systemic T cells [5]. Currently, the oral vaccination route has the advantage that the vaccine is inexpensive and easy to administer without causing any stress to the fish [6,7]. Some results support that transgenic plants could serve as a
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promising candidate for the future development of vaccines. Therefore, there is a need to exploit plant biotechnology for cost-effective oral fish vaccine development in plants,
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particularly in edible crops. The application of plant biotechnological tools for fish vaccine development is important for aquaculture because fish vaccines must be produced at a low cost and easily scalable, making them accessible and affordable for not only the aquaculture industry worldwide but also for improving the conditions for small
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fish farmers in developing countries [8,9]. Therefore, oral vaccination with antigens included in the feed would be the ideal method of vaccine delivery to fish. The mature GCRV particle is composed of 7 proteins, VP1-VP7. Among the 7
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structural proteins, there are 5 protein components (VP1, VP2, VP3, VP4 and VP6) involved in forming the viral core, and two proteins, VP5 and VP7, involved in the outer
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capsid shell [10]. Based on a comparative study between GCRV and other strains, VP7 presented high similarity [2].VP7 was expressed in Escherichia coli, suggesting that VP7 could be used as a potential subunit vaccine against GCRV infection [1]. The VP7 protein was reported to be involved in viral infection and could be suitable for developing a subunit vaccine for the control of GCRV infection [11]. Furthermore, the homology of the VP7 gene among different GCRV strains was 99%, and the conservation was better
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than that of VP5 and VP6, so the VP7 vaccine could resist the attack of the GCRV strain to a certain degree [12]. Therefore, it is appropriate to select VP7 as the target vaccine for GCRV.
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Cholera toxin B subunit (CTB) is known to be a potent mucosal adjuvant; it is arranged in a pentameric ring and contains five receptor-binding pockets for high-avidity association with cellular membranes containing GM1 [13]. CTB has been used as a
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carrier and an adjuvant for fused antigen proteins in plant-based vaccines to stimulate the uptake and immune response upon feed-mediated oral immunization [7]. It is expected
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that the plant-produced CTB-VP7 fusion proteins could be efficiently taken up by a mucosal immune system, resulting in an increased immune response against GCRV. So far, there are few reports describing fish vaccine antigens expressed in edible crops by plastid engineering, although the technology for plastid engineering of edible crops such
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as lettuce, tomato, potato, etc. has been developed and used to express a number of foreign proteins [9]. The use of plants for the expression and delivery of CTB-VP7 is attractive for creating oral vaccines against GCRV in grass fish.
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In this study, we investigated a recombinant protein in which one CTB and one VP7 were driven by the ubiquitin promoter. Then, the potential recombinant CTB-VP7 fusion
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protein was evaluated for use as a vaccine against GCRV infection [14]. Our results showed that rice calli could highly express the fusion protein of CTB-VP7 and have biological activity, suggesting that it could possibly be used as an orally administered vaccine against GCRV in grass fish.
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2. Materials and methods 2.1. Synthesis and production of the VP7 recombinant protein The sequence coding for the VP7 protein from GCRV was synthesized according to
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the deposited sequences in GenBank (GenBank AF403396). The VP7 gene was inserted into BamH I sites in pET28a (Novagen, Darmstadt, Germany) to construct pET28a-VP7. For expression, the plasmids pET28a and pET28a-VP7 were transformed into BL21
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(DE3) cells and induced with 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside). For purification, the ProBondTM Purification System (Invitrogen, Waltham, MA, USA)
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was used according to the manufacturer’s instructions. The recombinant protein was finally eluted from the column with the same buffer containing 150 mM imidazole. The VP7 fusion protein (rVP7) concentration was determined by the Bradford assay [15]. Whole BL21 cell lysates before and after induction, supernatant and precipitate after
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sonicating, rVP7 were added equal volume of 2×SDS loading buffer, and boiled for 10.0 min in a boiling water bath, followed by separation on 12% SDS-PAGE and visualization
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by staining with Coomassie brilliant blue R-250.
2.2. Preparation of the polyclonal antibody and Western blot
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Before immunization, 0.5-1.0 ml of blood from Balb/c mice was collected as a
negative serum control. A polyclonal antibody against rVP7 was generated as we described previously[16]. Whole BL21 cell lysates and rVP7 were separated on SDS-PAGE and proteins were subsequently transferred to nitrocellulose membrane (Thermo Scientific, Waltham, MA, USA) using a Mini Tran-Blot® Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA) at 80V for 60 min. After blocking for 2h in
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5% skimmed milk in phosphate-buffered saline (PBS), membrane was incubated overnight at 4°C with anti-rVP7 mouse polyclonal antibody (1: 5000). The secondary antibody HRP-conjugated goat anti-rabbit IgG (Sangon, Shanghai, China) were
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employed to react with the primary antibody for 1 h at room temperature. The protein bands were then visualized using a SuperSignal West Dura solution Extended Duration
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Substrate (Thermo Scientific, Waltham, MA, USA).
2.3. Construction and expression of the CTB-VP7 fusion gene
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For the CTB-VP7 fusion gene, the CTB fragment and VP7 gene were combined with a flexible connection linker. The synthetic CTB-VP7 was modified based on plant-optimized codon usage and fused to a translation signal (the Kozak sequence, GCCACC) at the front of the start codon and the ER retention signal,
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SEKDEL(TCTGAGAAAGATGAGCTA), at the 3’-terminus. For subsequent vector manipulations, BamH I restriction sites were introduced at the 5’- and 3′-termini of the CTB-VP7 fragment. The DNA products were then inserted into the bacterial expression
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vector pET28a, and the correct clone bearing the CTB-VP7 fusion gene was confirmed by gene sequencing. Then the plasmid pET28a-CTB-VP7 was transformed into BL21 (DE3)
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cells. Expression, purification and Western blot of the fusion protein (rCTB-VP7) were performed according to the above instructions. rCTB-VP7 was subsequently eluted from the column with the same buffer containing 200 mM imidazole.
2.4. Rice plant transformation and regeneration Agrobacterium-mediated genetic transformation experiments were carried out using
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the EHA105 strain harboring pUbi-CTB-VP7. The rice cultivar ‘ZH11’ was used for genetic transformation. This was performed under the control of the maize ubiquitin-1
generated and grown as we described previously [17].
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(Ubi) promoter and nopaline synthase (nos) terminator. Transgenic rice calli were
2.5. Genomic PCR analysis of the insertion of the CTB-VP7 fusion gene in the plant
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genome
DNA insertion was confirmed by genomic PCR analysis. Rice genomic DNA was
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extracted from calli, and subsequent PCR analysis was performed as we described previously [15]. PCR was performed using ExTaq polymerase (Takara, Dalian, China) and two primers, VP7F and VP7R (VP7F; 5′-GCAGAACATCACTGACCTGTGTG-3′, VP7R; 5′- CATAGCTCATCTTTCTCAGAATC-3′). Regenerated nontransgenic rice
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plants were used as negative controls. Twenty-five cycles of PCR were performed, and the conditions were as follows: denaturation at 94°C for 1.0 min, annealing at 56°C for 1.0 min and extension at 72°C for 1.0 min. PCR products were detected by 1.5% agarose
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gel electrophoresis followed by ethidium bromide (EB) staining.
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2.6. RNA analysis
Total RNA was extracted from rice calli using the Plant RNA Purification Reagent
(Invitrogen, USA) according to the manufacturer’s instructions. Total RNA was incubated with RNase-free DNase I (Life Technology, Gaithersburg, USA) to eliminate contaminated genomic DNA before being reverse transcribed into cDNA using oligo d(T)18 primers and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). The
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expression of the CTB-VP7 fusion gene was determined using qRT-PCR. The 20 µl PCR reaction mixtures contained 1 µl of each primer (0.5 µM), 2 µl of template, 6 µl of H2O and 10 µl of 2×iQTM SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) on the
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Bio-Rad CFX96™ Real-Time System (Bio-Rad, Hercules, CA, USA). The PCR conditions involved an initial denaturation cycle of 95°C for 3 min, followed by 25 cycles of 95°C for 10 s, 55°C for 30 s, and plate read. Each sample was run in triplicate.
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Relative gene expression levels were determined by the comparative threshold cycle method (2-∆∆ C t) with β-actin used as the reference gene. Two PCRs were performed with combination
of
primers
(F1⁄R1)
as
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a
follows:
F1
(5’-
GAAGTACCAGGTAGCCAGCA-3’) and R1 (5’-TTGTGTACAGGGTAAGGCGT-3’). The β-actin gene was amplified using an Actin-F⁄Actin-R primer set (5′-GTC AGC AAC TGG GAT GAT ATG G-3′/5’-TCT TCC TTG CTC ATC CTG TCA G-3’) as an internal
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control to normalize the amount of rice callus RNA.
2.7. Crude protein extraction from nontransgenic and transgenic rice calli
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Approximately 100 mg of rice callus tissue was frozen in liquid nitrogen, ground into a fine powder, and resuspended in 200 µl of protein extraction buffer [PBST plus 10
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µg/ml leupeptin and 1.0 mM phenylmethylsulfonyl fluoride]. Samples were centrifuged at 14,000 rpm in a microcentrifuge for 10 min at 4°C. Total soluble protein (TSP) concentrations were determined in supernatants by the Bradford assay [16].
2.8. SDS-PAGE and Western blot analysis of CTB-VP7 protein in rice calli
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The crude protein was extracted from rice calli as previously described [17]. To assess the integrity of CTB-VP7, Western blotting was carried out using TSP plant extracts from WT (wild-type), VT (vector-transgenic), and transgenic plants, and 50 ng
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of pure rCTB-VP7 was used as a control. Samples were boiled for 10.0 min and resolved by SDS-PAGE. At the same time, two repeated experiments were performed, one for
performed according to the above instructions.
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staining and one for Western blotting, using an anti-rVP7 antibody (1: 5000) were
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2.9. ELISA and GM1 binding assay of the CTB-VP7 protein in rice calli
ELISA was performed to quantify the amount of CTB-VP7 in rice calli. The CTB-VP7 content in the crude extract and the ability to bind GM1 were determined using
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a ganglioside-dependent ELISA [18]. TSP extracts were coated on ELISA plates.
2.10. Purification of the CTB-VP7 protein from rice calli Equal amounts of rice callus tissue (20.0 g) from five independent lines was ground
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in liquid nitrogen and homogenized in 20 ml of protein extraction buffer. Purification of CTB-VP7 from the crude protein extraction of rice calli was carried out as described in
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Su et al. [19].
2.11. GM1- binding assay of the CTB-VP7 protein in bacteria and rice calli The concentrations of rCTB-VP7 and CTB-VP7 from prokaryotes and rice calli
were determined using the Bradford assay with a calibration curve base on BSA [20].
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GM1-ELISA was conducted to determine the affinity of rCTB-VP7 and CTB-VP7 for the GM1 receptor. The two proteins were quantified by ELISA with the rVP7 antibody to ensure that the amounts of the two proteins were consistent. The wells of
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plates were coated with 100 µl of GM1 (0.3 µg/ml) (Sigma-Aldrich, MO, USA). After washing, the wells were blocked with 200 µl of blocking buffer containing milk and then
the previously described methods for indirect ELISA.
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incubated for 2 h at 37°C. The remaining procedures were conducted in accordance with
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2.12. Assay of the biological activity of the CTB-VP7 fusion protein from rice calli Indirect ELISA was performed to check the biological activity of CTB-VP7 from rice calli at different temperatures. To test the relative binding affinity of CTB-VP7 with GM1 receptors, microtiter plates were coated with 100 µl of GM1 (0.3 µg/ml) and
3. Results
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incubated at 4℃, 25℃, 37℃, 42℃, 47℃, and 100℃ for 2 h individually.
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3.1. Detection of VP7 expressed in E. coli and its antibody E. coli strain BL21 transformed with pET28a-VP7 was induced with IPTG for 3 h. As
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expected, the molecular weight of the expressed protein was approximately 37.0 kDa, as determined by SDS-PAGE. After ultrasonic disruption, the specific protein could only be detected in inclusion bodies but not in the supernatant, suggesting that rVP7 is expressed mainly in inclusion bodies inside bacterial cells. rVP7 was purified through a Ni2+ affinity column and then used to prepare a mouse polyclonal antibody. The mouse antibody titer against rVP7 was higher than 1:100,000, as determined by ELISA. No cross-reaction was
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detected for the negative control, indicating that the rVP7 antibody was successfully prepared. Western blotting was carried out and showed that the positive signal for the
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polyclonal antibody recognizing the rVP7 was at the expected size of approximately 37.0 kDa. In contrast, the control contained no signal in the assay. The results suggested that the prepared antibody could be used to detect rVP7. Western blotting indicated that the
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expressed rVP7 was specifically recognized by the mouse anti-rVP7 polyclonal antibody
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(Fig. 1).
3.2. CTB-VP7 fusion gene expressed in E. coli
E. coli strain BL21 transformed with pET28a-CTB-VP7 was induced with IPTG for 3 h. After ultrasonic disruption, the specific protein could only be detected in inclusion
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bodies but not in the supernatant, suggesting that the rCTB-VP7 was expressed mainly in inclusion bodies inside bacterial cells. rCTB-VP7 was purified through a Ni2+ affinity column. A specific band of approximately 49.0 kDa representing the rCTB-VP7 could be
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detected by SDS-PAGE and Western blotting with a mouse anti-rVP7 antibody, indicating that rCTB-VP7 was correctly expressed in E. coli (Fig. 2). Western blotting
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indicated that the expressed rCTB-VP7 was specifically recognized by the mouse anti-rVP7 polyclonal antibody.
3.3. Transformation of rice calli with CTB-VP7 The Kozak sequence (GCCACC) in front of the start codon and an ER retention signal (SEKDEL) before the stop codon were applied to the CTB-VP7 fusion gene to
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improve the expression level of the recombinant protein in transgenic plants. The binary recombinant plasmid pUbi-CTB-VP7, which was designed to express CTB-VP7 under the control of a ubiquitin promoter, was constructed (Fig. 3a). These plasmids
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(pUbi-CTB-VP7, pUbi) were introduced into rice plants by Agrobacterium. tumefaciens-mediated gene transformation respectively. After infection with A. 34
antibiotic-resistant
rice
scutellum-derived/embryogenic calli in 40 days.
calli
proliferated
from
150
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tumefaciens,
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3.4. Detection of the CTB-VP7 gene in transgenic rice calli
Characterization of transgenic callus integration of CTB-VP7 into rice genomes was confirmed by genomic PCR, which showed amplification of a 1.2 kb fragment corresponding to the CTB-VP7 gene amplified from the vector. Of the 34 regenerated rice
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calli tested, 15 lines were positive for amplification (Fig. 3b). No amplification was obtained using non-transgenic rice lines.
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3.5. Detection of CTB-VP7 transcripts in transgenic rice calli To analyze CTB-VP7 transgene transcript levels, qRT-PCR was performed using two
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pairs of primers and RNA from rice calli. Primers were designed in such a way that one pair could detect the transcript levels in addition to whole CTB-VP7, facilitating the evaluation of two coordinating gene expression levels. Transcript levels were similar among transgenic lines with the exception of CTB-VP7 line 11, which has an expression level 1.25- to 2.91-fold higher than other lines. Thus, coordinated expression of the
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CTB-VP7 sequences was equally effective at the transcript level. The transcript levels in
3.6. Detection of the CTB-VP7 protein in transgenic rice calli
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CTB-VP7 line 11 was 2.91 that of line 26 (Fig. 4).
The expression level of the CTB-VP7 protein in transformed calli was determined via ELISA in accordance with the methods established previously [17]. The analysis showed
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that CTB-VP7 protein was expressed in the rice calli at levels ranging from 0.56 to 2.17%, an average of 1.54% of the total soluble protein. Western blot analysis showed
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that the heterologous protein contributed by CTB-VP7 had an apparent molecular weight of 45 kDa, which is slightly larger than the theoretical counterpart (43 kDa), suggesting that the recombinant protein had been processed in plant cells (Fig. 5). No corresponding band could be visualized in WT and VT plants. In addition, the recombinant protein was
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recognized specifically by the VP7 polyclonal antibody; thus, the band was recognized as CTB-VP7 expressed in rice calli.
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3.7. Binding assay of the plant-produced CTB-VP7 protein to GM1 GM1-ELISA was conducted to determine the affinity of CTB-VP7 for the GM1
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receptor. The crude protein extract with CTB-VP7 produced in rice calli demonstrated a strong affinity for GM1 but not for BSA. The crude protein extracts from WT, VT and PBS rice calli showed no or very weak affinity for GM1. Thus, the strong relative binding efficiency of CTB-VP7 in crude protein extract for GM1 indicated that the transgenic rice callus-derived CTB subunit interacted with GM1 (Fig. 6).
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3.8. Binding affinity of the CTB-VP7 fusion protein from rice calli and bacteria The function of the CTB-VP7 fusion protein was correlated with the ability to bind to GM1. The binding affinity of rCTB-VP7 and CTB-VP7 to GM1 was determined by
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ELISA. Within the linear range of binding at variable molar concentrations of GM1, the order of binding affinity was CTB-VP7> rCTB-VP7. Thus, CTB-VP7 showed a higher
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affinity towards GM1 than rCTB-VP7 (Fig. 7).
3.9. Temperature and binding affinity of the CTB-VP7 fusion protein
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The binding affinity of CTB-VP7 was determined by GM1-ELISA carried out at different temperatures, such as 4℃, 25℃, 37℃, 42℃, 47℃, and 100℃. Within the linear range of binding at the same concentrations of GM1, the order of binding affinity was 37℃>42℃>47℃>25℃>4℃>100℃. Thus, CTB-VP7 showed a higher affinity for GM1 at
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37℃ than at other temperatures. Maximum binding of CTB-VP7 to GM1 was reported to
(Fig. 8).
4. Discussion
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occur within 2 h at 37℃, and approximately half of the binding affinity remains at 25℃
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Oral vaccines have received increasing attention owing to their convenience and
effectiveness. In the case of developing a virus vaccine for grass fish, a plant-based vaccine is a good choice [5]. Compared with bacteria, plants are more able to express viral proteins because of their glycosylation process. This is very important because glycan moieties are an important factor in the generation of neutralizing antibodies. The recombinant proteins produced in plants have higher immunogenicity when administered
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with the plant cell wall components [21]. Fish culture in paddy fields has a long history and ecological significance in China. Furthermore, rice is a model plant, and the technology of genetic transformation is mature and convenient. The use of rice calli as
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bioreactors is more feasible because it does not involve assessing the ecological safety of transgenic plants. Therefore, the use of rice callus cells to produce oral GCRV vaccines is a better choice.
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The present study aimed to identify the genetic engineering vaccine potential of the outer shell protein VP7. As the outer shell protein, VP7 might have key effects on viral
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infection [22]. The fact that anti-VP7 polyclonal antibodies efficiently blocked viral infection here supported that VP7 was involved in viral infection and may be a good candidate for the development of a vaccine. Meanwhile, CTB has been widely used to facilitate antigen delivery by serving as an effective mucosal carrier molecule for
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chemically or genetically conjugated autoantigens for the induction of oral tolerance [23,24]. Conjugation with CTB may greatly facilitate antigen delivery and presentation to the gut-associated lymphoid tissues (GALT) due to its affinity for the cell surface
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receptor GM1 located on cells of the GALT, including the membranous cells and enterocytes, for increased uptake and immunologic recognition [25]. Therefore, the
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CTB-VP7 fusion protein could generate protective serum and mucosal antibodies, resulting in an increased protective immune response of grass carp against GCRV infection.
The genome of transgenic rice calli was analyzed for genetic stability by PCR
analysis. It was confirmed that the CTB-VP7 coding sequence was successfully integrated into the genome of rice. Moreover, the results of the qRT-PCR detection of CTB-VP7
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showed that it was expressed in transgenic rice. The expression of the CTB-VP7 protein in different transgenic plants showed wide variation. This is consistent with the transcript level determined by real-time PCR [26]. The variation in expression is likely due to the
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integration of the transgene at different positions in the genomes of independent transgenic lines [27]. These different expression levels of the CTB-VP7 fusion gene among the transgenic plants are due to the different incorporation sites of the target gene
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in the chromosomes of individual plants, known as the ‘position effect’[28]. Our results indicated that the transgenic rice calli was stable enough to maintain its genetic integrity.
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The integrity of the CTB-VP7 protein was confirmed by Western blot analysis using an anti-rVP7 antibody. This further confirmed that the rice-callus-derived fusion protein retained its antigenic properties and displayed the antigenic determinants of both CTB and VP7. We successfully expressed CTB-VP7 in rice calli using an A.
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tumefaciens-mediated transformation method. In addition, the biological activity of the plant-produced CTB-VP7 for GM1 was confirmed by GM1-ELISA. However, the molecular mass of CTB-VP7 expressed in rice calli was approximately 45 kDa, which is
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smaller than the 6His-CTB-VP7 fusion protein (49 kDa) and slightly larger than its theoretical counterpart (43 kDa), suggesting that the recombinant protein had been
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processed in rice calli through processes such as glycosylation and phosphorylation [25]. This is in line with the major advantages of plant cells for their ability to provide eukaryotic posttranslational modifications and expression products that possess high bioactivity and immunogenicity. The crude extracts from the CTB-VP7 plant tissues were found to have biology activity based on an in vitro assay using GM1[29]. The nuclear-encoded CTB-VP7
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protein retains the pentameric structure of CTB as it binds to GM1 and is recognized by rVP7-specific antibodies. This is the first study describing the production of biologically active fish CTB-VP7 using transgenic plants. In light of the apparent aggregation of the
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two proteins, it may be useful to increase the size of the intermolecular linker (Gly4Ser)3 to further reduce steric hindrance to facilitate assembly of the fusion protein monomers into pentamers rather than oligomers of variable size [30]. The linker was designed to
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direct the protein folding [31]. In general, the antigenic activity of CTB-VP7 suggests that it was correctly assembled and functionally produced in the plant cell.
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Immunoblot analysis and GM1-ELISA experiments showed the probability that monomeric B subunits accumulate within the lumen of the ER of plant cells where self-assembly into oligomeric, possibly pentameric, GM1 bound forms occur [32]. Therefore, these results indicated that the extension of CTB plus a linker to VP7 did not
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prevent CTB pentamer formation and that both components of the fusion protein retained antigenicity. The binding assay showed that a pentameric CTB was formed with functionally correct folding and assembly of the chimeric CTB-VP7 expressed in rice
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calli [33]. The plant-synthesized CTB-VP7 shows a higher affinity for binding to GM1 compared to the bacterial rCTB-VP7. The glycosylation of the plant-expressed CTB-VP7
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was predicted to facilitate functionally more favorable folding of the CTB-VP7 in bacterial cells [34]. The chaperones, other folding enzymes and glycosylation in plant cells may lead to an increase in the affinity of CTB-VP7 for GM1 receptors compared to the nonglycosylated bacterial rCTB-VP7. The glycosylation of the plant-expressed CTB was predicted to facilitate functionally more favorable folding of the CTB in rice cell [35,36]. The plant-expressed
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CTB-VP7 bonded to GM1 with different affinities at different temperatures. Maximum binding of the cholera toxin to GM1 was found to occur at 37℃. These results indicated that temperature affects the spatial structure of the plant-expressed CTB-VP7 fusion
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protein, especially the configuration of CTB, and thus affects the binding power of the protein to GM1. Fishes live in water, and therefore, the temperature of the water has a strong influence on the physiological function of fish [37]. The suitable temperature
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range for fish growth is 15-30℃[38]. Within this temperature range, the binding of CTB-VP7 from rice calli to GM1 still has activity, but its activity is lower than that at
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37℃. Therefore, CTB-VP7 can be used as an oral vaccine for fish.
5. Conclusions
This study demonstrated that a GCRV VP7 capsid antigen coupled with CTB could
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be synthesized and assembled into a functional form in rice cells as a step towards developing an oral plant-based vaccine for grass fish to prevent GCRV. The strong GM1 binding affinity of the CTB-VP7 fusion protein suggests its potential as an oral vaccine,
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and future studies will address its immunogenic potential in grass fish. Our results provide a reliable system for further in vitro expression and assembly of GCRV particles
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using a plant expression system.
Conflicts of interest
The authors declare that they have no competing interests.
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Acknowledgments This work was supported by the Shandong Provincial Natural Science Foundation, China (No. ZR2016CM10), Key R & D program of Shandong Province, China (No.
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2018GSF121035) and A Project of Shandong Province Higher Educational Science and
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Technology, China (No. J13LE09)
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Figure Legends
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Fig1. VP7 fusion protein prokaryotic expression vector construction, expression, purification and test a,VP7 prokaryotic expression vector construction; M, DNA marker; 1,VP7 prokaryotic expression vector digested with BamHI; b, VP7-fusion prokaryotic expression; M, protein marker; 1,the no-induced germ total proteins; 2, the induced germ total proteins; C, Purification of rVP7; M, protein marker; 1, Supernatant after sonicating and centrifuging; 2, precipitate after sonicating and centrifuging; 3, purified rVP7, d, Western blot of the antibody from
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rVP7.
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rVP7. M, protein marker; 1, no-induced germ total proteins, 2, the purified rVP7; Arrows in b, c and d indicate
Fig2. CTB-VP7 fusion protein prokaryotic expression vector construction, expression, purification and test
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ACCEPTED MANUSCRIPT a, Schematic structure of the fusion gene CTB-VP7.CTB-VP7construct was engineered with a flexible peptide
linker between CTB and VP7; b, CTB-VP7 prokaryotic expression vector construction. M, DNA marker. 1, The
integration fusion gene fragment with the VP7 and CTB. 2,CTB-VP7prokaryotic expression vector digested with
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BamHI; c, CTB-VP7 fusion prokaryotic expression. M, Protein marker. 1, no-induced germ total proteins. 2,
induced germ total proteins. 3, Supernatant after sonicating and centrifuging. 4, Precipitate after sonicating and
centrifuging; d, SDS-PAGE of rVP7 and rCTB-VP7. M, protein marker. 1, rVP7. 2, rCTB-VP7; e, Western blot of
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rVP7 and rCTB-VP7 by rVP7 antibody. M, protein marker; 1, rVP7; 2, rCTB-VP7. Arrows in c ,d and e indicate
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rCTB-VP7.
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Fig 3. a: Schematic representation of designed constructs for expression of the CTB-VP7 gene in rice calli.
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HYG, Hygromycin B, was used for screening the transformants; the CTB-VP7 gene was driven by the Ubi promoter and terminated by the Nos terminator. LB and RB represent left and right borders respectively. b: Genomic DNA PCR analysis used to detect the CTB-VP7 gene in transgenic rice callus lines. M, DL2000 molecular markers; C, PCR control; VT, transformed rice calli with empty-vector; WT, nontransgenic rice calli; 4, 9, 11, 12, 15, 17, 26, different transgenic rice callus lines.
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Fig 4. The expression levels of CTB-VP7 by quantitative real-time PCR
The accumulation of CTB-VP7 transcripts was determined by qRT-PCR. Total RNA was isolated from
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rice calli of WT (wild type), VT (transformed rice calli with empty-vector) and transgenic rice callus lines (4, 11, 15, 17, 26). The constitutively expressed β-actin mRNA, set to 100%, was used as an internal standard to normalize relative amounts of transcripts in all experiments. The mRNA samples from five independently harvested rice callus lines were analyzed. Data showed the means±SE of five
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independent experiments.
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ACCEPTED MANUSCRIPT Fig 5. Total soluble protein extracts were resolved by SDS-PAGE under denaturing condition and subjected
to blotting and immunodection by anti-rVP7 serum. a: SDS-PAGE of crude protein extracted from the rice calli of empty vector, transgenic vector. b: Western blot examination of CTB-VP7 in different rice callus lines.
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26, different rice callus lines. Arrows in a and b indicate CTB-VP7.
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M, protein marker. C0, CTB-VP7 fusion protein; VT, transformed rice calli with empty-vector; 4, 11, 15, 17 and
Fig 6. GM1 binding CTB-VP7 from rice calli by ELISA
ELISA assay was performed by coating the wells of the microtiter plate with GM1 or BSA. Anti-rVP7
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antibody was added to identify the biological binding activity of plant-derived CTB-VP7 to GM1.
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WT, wild type rice calli; VT, transformed rice calli with empty-vector; 4, 11, 15, 17 and 26, transformed rice calli with CTB-VP7. Data are expressed as mean±SE. Error bars represent standard error of the mean.
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Fig 7. GM1 binding test of CTB-VP7 fusion protein from bacterial and rice calli
The purification of CTB-VP7 fusion protein from bacterial and rice calli, BSA as control, were serially diluted
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from 1 (1:20), 2 (1:50), 3 (1:100), 4 (1:200) to 5 (1:400) and incubated with anti-rVP7 antibody.
Fig 8. GM1 binding test of the CTB-VP7 fusion protein from rice calli under different temperatures
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The purification of CTB-VP7 fusion protein from rice calli, BSA as control, were serially diluted from 1 (1:20), 2 (1:50), 3 (1:100), 4 (1:200) to 5 (1:400) and incubated with anti-rVP7 antibody.
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