Expression of avian reovirus σC protein in transgenic plants

Journal of Virological Methods 134 (2006) 217–222

Expression of avian reovirus ␴C protein in transgenic plants Liang-Kai Huang a , Sin-Chung Liao b , Ching-Chun Chang a,∗ , Hung-Jen Liu c,∗ a

Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan Department of Biological Science and Technology, Meiho Institute of Technology, Pingtung, Taiwan c Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung, Taiwan b

Received 4 October 2005; received in revised form 12 January 2006; accepted 12 January 2006 Available online 20 February 2006

Abstract Avian reovirus (ARV) structural protein, ␴C encoded by S1 genome segment, is the prime candidate to become a vaccine against ARV infection. Two plant nuclear expression vectors with expression of ␴C-encoding gene driven by CaMV 35S promoter and rice actin promoter were constructed, respectively. Agrobacterium containing the S1 expression constructs were used to transform alfalfa, and transformants were selected using hygromysin. The integration of S1 transgene in alfalfa chromosome was confirmed by PCR and histochemical GUS staining. Western blot analysis using antiserum against ␴C was carried out to determine the expression of ␴C protein in transgenic alfalfa cells. The highest expression levels of ␴C protein in the cellular extracts of selected p35S-S1 and pAct1-S1 transgenic alfalfa lines were 0.008% and 0.007% of the total soluble protein, respectively. The transgenic alfalfa cells with expression of ␴C protein pave the way for the development of edible vaccine. © 2006 Elsevier B.V. All rights reserved. Keywords: Alfalfa cells; Avian reovirus; ␴C protein

1. Introduction Avian reoviruses (ARV) are important poultry pathogens that cause a number of avian diseases, including viral arthritis, malnutrition syndrome, and chronic respiratory disease. The genome of ARV consists of 10 segments of double stranded RNA which encoded for at least 12 distinct proteins (Varela and Benavente, 1994; Liu et al., 2003). ␴C, encoded by the S1 genome segment, is one of the outer capsid proteins, with a molecular weight ranging from 35 to 39 kDa, depending on the virus strains. It is a cell attachment protein involved in induction of apoptosis (Shih et al., 2004). Although ␴C is a minor capsid protein, it is one of the major antigens responsible for inducing the neutralizing antibodies which may protect host cells from the viral infection (Wickramasinghe et al., 1993). Therefore, ␴C has become one of the major candidates to develop a subunit vaccine against ARV infection. ARV ␴C protein has been successfully expressed in Escherichia coli (Shapouri et al., 1995; Liu et al., 2002), and

∗

Corresponding authors. E-mail addresses: [email protected] (C.-C. Chang), [email protected] (H.-J. Liu). 0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.01.013

recently in baculovirus/insect cells expression systems (Hu et al., 2002). Plant-based edible vaccines have several potential advantages over traditional vaccines. They are cheap, safe and scale-up rapidly. Additionally, they are easy to store and deliver orally, reduce the cost of syringes and needles and eliminate injection anxiety (Streatfield and Howard, 2003). Human hepatitis B surface antigen (HBsAg) was the first subunit vaccine to be successfully expressed in tobacco plants (Mason et al., 1992). HBsAg forms virus-like particles (VLP) of 22 nm in average size in tobacco leaves. They resemble the VLP particles produced in Saccharomyces cerevisiae, used in the current hepatitis B vaccine. In addition, an injection of purified plant-expressed HBSAg provoked an antibody response similar to that induced by commercial yeast-derived vaccine (Thanavala et al., 1995). Oral delivery of potato expressing with HBSAg in mice showed a stimulation of serum IgG that could be greatly boosted with a single sub-optimal dose of yeast-derived vaccine (Richter et al., 2000; Kong et al., 2001), which demonstrated great promise for oral delivery of a plant-based vaccine. Small scale human clinical trials in which the antigen has been delivered orally in potato indicate its safety and immunogenicity (Thanavala et al., 2005). To date, the surface antigens of a variety of human pathogens have been successfully expressed in plants

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(Streatfield and Howard, 2003), and immunity has been induced in animal model many times. Furthermore, multiple antigens from different pathogens can be simultaneously expressed in the same plants to create a plant-based multicomponent vaccine for simultaneous protection against multiple diseases (Yu and Langridge, 2001). Development of an edible vaccine for livestock is also currently a focus of research. For instance, the VP1 antigen of foot and mouth disease virus has been expressed in transgenic Arabidopsis (Carrillo et al., 1998), alfalfa (Wigdorovitz et al., 1999; Dus Santos et al., 2002) and potato (Carrillo et al., 2001) with effective protection has been demonstrated in mice (Wigdorovitz et al., 1999). The glycoprotein S of swine transmissible gastroenteritis virus (TGEV) has been expressed in Arabidopsis (Gomez et al., 1998), potato (Gomez et al., 2000), tobacco (Tuboly et al., 2000) and maize (Lamphear et al., 2002) with resultant recombinant plant proteins that can boost lactogenic immunity by oral delivery (Lamphear et al., 2004). The major capsid protein VP6 of the bovine group A rotavirus has been also expressed in potato tuber (Matsumura et al., 2002). Expression of the VP4 protein of bovine rotavirus (BRV) in transgenic alfalfa was effective in inducing an anti-rotavirus antibody response in adult female mice and inducing lactogenic immunity in newborn mice against a virulent rotavirus (Wigdorovitz et al., 2004). The glycoprotein E2 of bovine viral diarrhea virus has been successfully expressed in transgenic alfalfa (Dus Santos and Wigdorovitz, 2005). The VP2 protein of chicken infectious bursal disease virus (IBDV) has been expressed in Arabidopsis (Wu et al., 2004a) and the efficacy of plant-produced VP2 as a vaccine against IBD has been demonstrated (Wu et al., 2004b). The glycoproteins F and HN of Newcastle disease virus (NDV) were correctly expressed in potato, and induced anti-NDV specific antibodies in mice (Berinstein et al., 2005). These examples open the way for the development of an edible vaccine against pathogen infection in livestock. To investigate the possibility of developing an edible vaccine against ARV infection for the poultry industry, we first expressed the ␴C protein of ARV in alfalfa cells.

Fig. 1. Schematic representation of p35S-S1 and pAct1-S1 expression constructs. The backbone plasmid is pCAMBIA 1301. S1, Avian reovirus S1 cDNA fragment; 35Sp, cauliflower mosaic virus 35S promoter; Act1p, rice actin1 promoter; HygR, Hygromycin B resistance gene; Gus, ␤-glucuronidase gene; PolyA, CaMV polyadenylation signal; Nos-t, nopaline synthase gene terminator; LB, left border; RB, right border.

Act1-Sma (5 -CCCGGGCTCGAGGTCATTCATATGC-3 ) and Act1-Hind (5 -AAGCTTCTTCTACCTACAAAAAAAGCTC3 ), and were cloned into pGEM-T vector to generate pTA-Act1. A 1253-bp fragment of PstI/HindIII after digestion with pTAAct1 and 1248 bp fragment of KpnI/HindIII from pS1-BSK were ligated with the 11822-bp fragment of PstI/KpnI digested from pCAMBIA1301 to generate pAct1-S1 (Fig. 1). The pAct1-S1 and p35S-S1 plasmid DNA were checked by restriction analysis, and then transformed individually into Agrobacterium tumefaciens strain LBA4404 by electroporation. 2.2. Plant transformation

2. Materials and methods

Alfalfa (Medicago sativa L.) seeds were germinated on Murashige and Skoog (MS) media, and grown to 5–7 weeks old. Petiole explants of alfalfa seedlings were cut into 0.5–1 cm sections and incubated with Agrobacteria tumefaciens LBA4404 carrying the binary vector pAct1-S1 or p35S-S1 with suction for 10 min. The explants were co-cultivated for 4 days in the dark on SH induction medium (McKersie et al., 1999) and then were washed two times, each time with half-strength MS solution containing 500 ␮g cefotaxime/ml for 1 h. After washing, the explants were kept in the same induction medium containing both 500 ␮g cefotaxime/ml and 50 ␮g hygromycin/ml for selection and fresh medium was supplied every 2 weeks.

2.1. Construction of σC plant expression cassette

2.3. PCR analysis

The S1 cDNA encoding ARV S1133 ␴C protein was cloned as described previously (Liu et al., 1999). The S1 cDNA was amplified by PCR using a pair of primers with forward primer S1-BstE (5 -GGTCACCTTAGGTGTCGATGCCGGTA-3 ) and reverse primer S1-Hind (5 -AAGCTTACCATGGCGGGTCTCAATCC-3 ). Nine hundred and eighty-one base pair fragment of S1 gene PCR product was cloned into pGEM-T vector (Promega, USA) to generate pTA-S1. A 981-bp fragment of BstEII/HindIII after digestion with pTA-S1 was ligated with the 3739 bp fragment of BstEII/HindIII from pHSP101 (Lu, 2004) to generate pS1-BSK. A 1809 bp fragment of EcoRI/KpnI after digestion with pS1-BSK were ligated with the 11,837 bp fragment of EcoRI/KpnI from pCAMBIA1301 (CAMBIA, Australia) to generate p35S-S1 (Fig. 1). The promoter sequence of rice actin1 gene were PCR amplified with a primer pair of

DNA was extracted from alfalfa transformants as described previously by McKersie et al. (1999). PCR was performed with specific primers for S1 gene (S1-BstE and S1-Hind) on a 5 ␮l DNA solution in a Thermal Cycler (Hybaid, UK) using 200 pmol of each primer, 200 nM of each deoxyribonucleotide triphosphate, 3 units of Taq DNA polymerase, and 5 ␮l of 10× Taq DNA polymerase buffer in a 50 ␮l reaction mixture. The amplification was started with one 2 min cycle at 94 ◦ C followed by 35 cycles of 1.5 min at 94 ◦ C, 2 min at 60 ◦ C, and 3 min at 72 ◦ C, and this was followed by one 5 min cycle at 72 ◦ C. Each PCR sample (30 ␮l) was electrophoresed on a 1% agarose gel and visualized by staining with ethidium bromide. The sequences of the PCR primers used for analysis of the introduced S1 gene were S1-BstE and S1-Hind as described in previous section. The expected size of PCR products is about 981 bp in length.

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2.4. β-Glucuronidase (GUS) assays Expression of GUS in alfalfa transformants was assayed by histochemical staining. Alfalfa callus were incubated in 5-bromo-4-chloro-3-indolyl-␤-d-glucuronide (X-gluc) solution containing 0.1 M phosphate buffer, 1 mg X-gluc/ml, 0.05 mM potassium ferricyanide, 0.05 mM potassium ferrocyanide and 0.1% Triton X-100. The reaction mixture was incubated overnight at 37 ◦ C, and samples were stored in 75% (v/v) ethanol. 2.5. Polyclonal antibody preparation and immunoblotting The DNA encoding the 326 amino acids from S1 were amplified by PCR with a primer pair of forward primer S1-Nco (5 CCATGGCGGGTCTCAATCCATC-3 ) and reverse primer S1Hind (5 -AAGCTTGGTGTCGATGCCGGTACG-3 ) and were cloned into pGEM-T vector to generate pTA-S1Ab. A 981bp fragment of NcoI/HindIII digested from pTA-S1Ab were inserted into the NcoI/HindIII site of expression vector pET42a (Invitrogen, CA), which generates a C-terminal histidinetagged fusion protein in E. coli BL21 (DE3). This protein was overexpressed, purified using a Ni-nitrilo-tri-acetic acid agarose resin (Invitrogen, USA), and used to immunize rabbits. Polyclonal antiserum was obtained and used directly in immunoblotting experiments. Total soluble proteins were extracted from transgenic and untransformed alfalfa cells with protein extraction buffer (50 mM Tris–HCl pH 8.0, 10% sucrose, 1% ␤mercaptoethanol, 1% DMSO, 0.2% Triton X-100, 0.3 M NaCl, 1.75 mg ascorbic acid/ml, 0.5 mg PMSF/ml). Protein concentrations were determined by using the Bradford method (Bio-Rad, USA). Equal mass amounts of proteins (10 ␮g) from different transgenic lines were separated in 10–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blocking was performed in 1× TBST (10 mM Tris pH 7.5, 150 mM NaCl, and 0.05% Tween-20) containing 5% nonfat dry milk. Blots were incubated with a primary antibody against ␴C (1–10,000 dilution) or monoclonal antibody against HSP70 (1–1000 dilution) (Stress Gene, USA) in 1xTBST at room temperature for 1 h. They were then washed three times with 1XTBST, and reactive bands were detected using an alkaline phosphate-conjugated secondary antibody. To quantitate the expression levels of ␴C

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protein in alfalfa cellular total soluble proteins, a known amount of bacteria-expressed ␴C fusion protein (0.1, 0.25, 0.5 and 5 ng) was electrophoresed in the same gel and immunodetected to establish a standard curve. Signals were scanned with a densitometer and quantified by ImageQuant software. 2.6. Assessment of oligomerization capacity of σC protein To test the multimerization capacity of ␴C protein, cellular proteins from transgenic and untransformed alfalfa cells were performed under both dissociating and nondissociating conditions. Samples were incubated in Laemmli sample buffer at either 30 ◦ C for 30 min to retain maximal oligomerization or at 90 ◦ C for 5 min to denature oligomer (Shih et al., 2004). Subsequent analysis of the samples by 10–20% SDS-PAGE was followed by transfer to PVDF membranes, and the reactive protein was detected with anti-␴C serum as described previously. 3. Results and discussion 3.1. Construction of S1 plant expression cassettes Two plant S1 gene expression cassettes were constructed based on the backbone plasmid of pCAMBIA1301. Expression constructs of p35S-S1 and pAct1-S1 are the S1 gene expression driven by the cauliflower mosaic virus (CaMV) 35S promoter and the rice actin1 promoter, respectively (Fig. 1). In addition to the S1 expression cassette, hygromycin B resistant gene and Gus reporter gene expression cassettes are present within the T-DNA region for each expression construct. Transformation of alfalfa explants with A. tumefaciens LBA 4404 carrying p35S-S1 or pAct1-S1 binary vector was carried out, and then transformants were selected in induction medium containing 100 ␮g hygromycin/ml. Thirty-nine and 29 hygromycinresistant transformants were selected from p35S-S1 and pAct1S1 transformed alfalfa cells, respectively. GUS histochemical staining was assayed to rapidly confirm the integration of TDNA into alfalfa chromosome. Transgenic lines of 5, 11, 12, 24, 56, and 62 from p35S-S1 expressed alfalfa cells and transgenic lines of 2, 8, 10, 20, 35 and 38 from pAct1-S1 expressed alfalfa cells were stained blue (Fig. 2). This result suggested

Fig. 2. Histochemical GUS staining of alfalfa transformants. Transformants of hygromycin resistant alfalfa cells from p35S-S1 (A) and pAct1-S1 (B) expression lines were stained with X-gluc solution. Six lines of each p35S-S1 and pAct1-S1 expressed alfalfa cells were stained blue, which indicated the expression of GUS proteins. Number indicates different lines of transformed alfalfa cells. W, untransformed alfalfa cells. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 3. PCR analysis of S1 transformants. Transformants of hygromysin resistant alfalfa cell lines were PCR amplified to confirm the integration of S1 gene in chromosome. Twenty-three lines of p35S-S1 (left panel) and 21 lines of pAct1-S1 (right panel) expressed alfalfa cells were PCR positive, and only 14 transformants from each expression construct were shown. Number indicates different transformed alfalfa lines. M, 1 kb marker; W, untransformed alfalfa; P, p35S-S1 or pAct1-S1 plasmid DNA. The 981 bp of PCR products were expected.

the expression of ␤-glucuronidase in those selected transgenic alfalfa lines. The integration of S1 gene into the chromosome of alfalfa cells was further checked by PCR assay with a primer pair from S1 gene, and 981 bp was the expected size. Twentythree out of 39 transformants from p35S-S1 expression lines, and 21 out of 29 transformants from pAct1-S1 expression lines were PCR positive, and contained the S1 transgene in the chromosome (Fig. 3). All the GUS positive alfalfa cells contained the S1 gene integration in their chromosomes. However, alfalfa cells with S1 gene integration did not necessarily express GUS. Since S1 and Gus genes are present in the separated expression cassette, expression of GUS proteins may be silenced in most of transgenic alfalfa lines. Nevertheless, the histochemical GUS assay provides a convenient way to identify transformants with T-DNA integration. 3.2. Expression of σC protein in alfalfa cells Total soluble proteins (TSP) were extracted from transgenic and untransformed alfalfa cells, and separated in 10–20% gradient SDS-polyacrylamide gels. Western blot analysis using antiserum against ␴C protein demonstrated the presence of the corresponding 35 kDa protein in fourteen transgenic alfalfa cell lines of p35S-S1 and in eight transgenic alfalfa cell lines of pAct1-S1, but was absent in untransformed plants (Fig. 4A and C). The ␴C protein expression level was quantitated and compared among the transgenic alfalfa cell lines with normalization of HSP70 proteins (Fig. 4B and D). The highest ␴C protein level in the p35S-S1 expression lines (line 21) and pAct1S1 expression lines (line12) can reach 0.008% and 0.007% of total soluble proteins, respectively. The average expression levels of ␴C protein from the 14 lines of p35S-S1 and 8 lines of pAct1-S1 transgenic alfalfa cells were 0.0046% and 0.0049% of total soluble proteins, respectively. Three transgenic alfalfa cell lines 21, 57 and 58 were expressed significantly higher than the other 11 transgenic lines in p35S-S1, with an average of 0.0077% of total soluble proteins (Fig. 4B). Four transgenic alfalfa cell lines 1, 2, 12 and 16 were expressed significantly higher than the other four transgenic lines in pAct1-S1, with an average of 0.0068% of total soluble proteins (Fig. 4D). This result suggested no significant difference in the regulation of ␴C protein expression between 35S and the actin pro-

moter. In addition, not all GUS positive alfalfa cells can be detected the expression of ␴C protein, suggesting that gene silencing had occurred in S1 for some GUS positive alfalfa lines. We encountered low levels of ␴C antigen expressed in alfalfa cells, which is also one of the major hurdles for the development of plant-based vaccine as reported in other systems (Mason et al., 1992; Matsumura et al., 2002; Streatfield and Howard, 2003). The low levels of foreign protein expression in plants may be due to a variety of factors affecting transcription, translation, and stability of the RNA transcripts and proteins. In this study, two different strong constitutive promoters were used to drive S1 gene expression, however, neither one significantly increased ␴C protein levels in transgenic alfalfa cells, suggesting that steps other than transcription level are major obstacle to its overexpression. Codon usage is one of the factors which may cause the low expression levels of ␴C protein, since we found that ACG (Thr), GCG (Ala) and TCG (Ser) are commonly used codons in the S1 genome segment, but they are rarely used in the nuclear genes of alfalfa cells. Previous study also has been demonstrated that protein expression levels of synthetic neutralizing epitope gene of porcine epidemic diarrhea virus (PEDV) with optimization of codon usages was much higher than that of native gene in transgenic tobacco plants (Kang et al., 2005). Currently, in order to greatly enhance ␴C protein expression level in alfalfa, we are synthesizing the S1 gene to optimize the alfalfa codon usage. 3.3. Assessment of oligomerization capacity of σC protein in alfalfa cells ␴C protein forms homotrimer in its native state and binds specifically to avian cells (Martinez-Costas et al., 1997; Grande et al., 2000). To investigate if ␴C protein is present in a mutimer in alfalfa cells, we either boiled one group of the protein and one was not before separating them in SDS-PAGE. A protein band with a molecular mass of 35 kDa was present in the boiled sample from transgenic alfalfa cells that was not present in the untransformed cells, indicating that this is the molecular mass of monomeric ␴C (data not shown). This band was also observed in a sample that was incubated at 37 ◦ C. However, no visible band with a molecular mass of 100 kDa corresponding to ␴C trimer

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Fig. 4. Immunoblot analysis of S1 transgenic alfalfa lines. Ten micrograms of total soluble proteins from transgenic and untransformed alfalfa cells were separated in a 10–20% SDS-polyacrylamide gel, transferred to a PVDF membrane, and subjected to western blot analysis with anti-␴C or anti-HSP70 serum. Expression of ␴C protein was detected in 12 lines of p35S-S1 (A) and 8 lines of pAct1-S1 (C) transformed alfalfa cells. The expression level of ␴C protein in p35S-S1 (B) and pAct1-S1 (D) lines of alfalfa cells were quantitated based on known amount of purified bacteria-expressed ␴C fusion protein as standard, and using Hsp70 as internal control to normalize different transgenic lines. Number indicates different transgenic alfalfa lines. W, untransformed alfalfa.

as previously reported (Grande et al., 2000) was detected. Our result suggested that ␴C protein is present in a monomer state in alfalfa cells. 4. Conclusion Transgenic plants offer a novel and safe method of vaccine development. Future demonstration of the efficacy of ␴C protein expressed in alfalfa cells to prevent ARV infection will further strengthen the concept of edible vaccine for control of poultry diseases.

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