Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Journal Pre-proof Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Sa Dong, Meijing Gao, Zongyi Bo, Lingjun Guan, Xiaodan Hu, Hanxiaoya Zhang, Beibei Liu, Pan Li, Kangli He, Xianjin Liu, Cunzheng Zhang PII:

S0141-8130(19)38326-6

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.152

Reference:

BIOMAC 14456

To appear in:

International Journal of Biological Macromolecules

Received date:

15 October 2019

Revised date:

5 December 2019

Accepted date:

15 January 2020

Please cite this article as: S. Dong, M. Gao, Z. Bo, et al., Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.01.152

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© 2018 Published by Elsevier.

Journal Pre-proof Title Page Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody

Authors: Sa Dong a,b,1, Meijing Gao a,1, Zongyi Bo c,1, Lingjun Guan b, Xiaodan Hu a, Hanxiaoya Zhang a, Beibei Liu a, Pan Li a, Kangli He b, Xianjin Liu a, Cunzheng Zhang a,*

Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding Base, Key

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a

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Laboratory of Control Technology and Standard for Agro-product Safety and Quality, Ministry of Agriculture, Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences,

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210014 Nanjing, PR China

College of Horticulture and Plant Protection, Yangzhou University, 225009 Yangzhou, PR China

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College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu 210095, People’s

Corresponding author.

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*

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Republic of China

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b

E-mail address: [email protected] (C. Zhang). These authors contributed equally to this work.

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Abstract: There are plenty of applications of Cry1A toxins (Cry1Aa, Cry1Ab, Cry1Ac) in genetically modified crops, and it is necessary to establish corresponding detection methods. In this study, a single-chain variable fragment (scFv) with high affinities to Cry1A toxins was produced. First, the variable regions of heavy (VH) and light chain (VL) were amplified from hybridoma cell 5B5 which secrete anti-Cry1A monoclonal antibody (mAb) and then spliced into scFv-5B5 by overlap extension polymerase chain reaction (SOE-PCR). Subsequently, site-saturation mutagenesis was performed after homology modeling and molecular docking, which showed that asparagine35, phenylalanine36,

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isoleucine104, tyrosine105, and serine196, respectively, located in VH complementarity-determining region (CDR1 and CDR3) and VL framework region (FR3) were key amino acid sites. Then, the mutagenesis

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scFv library (1.35×105 CFU/mL) was constructed and a mutant scFv-2G12 with equilibrium

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dissociation constant (KD) value of 9.819×10-9 M against Cry1Ab toxin, which was lower than scFv-5B5 (2.025×10-8 M) was obtained by biopanning. Then, enzyme-linked immunosorbent assay

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(ELISA) was established with limit of detection (LOD) and limit of quantitation (LOQ) of 4.6-9.2 and

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11.1-17.1 ng mL−1 respectively for scFv-2G12, which were lower than scFv-5B5 (12.4-22.0 and

Cry1A toxins.

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23.6-39.7 ng mL−1). Results indicated the promising prospect of scFv-2G12 used for the detection of

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Keywords: Cry1A toxins; ScFv antibody; Site-saturation mutagenesis

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1. Introduction Cry1A toxins (Cry1Aa, Cry1Ab and Cry1Ac) are secreted by bacteria Bacillus thuringiensis (Bt) which are known to be able to kill Lepidoptera and Coleopteran pests effectively and harmless to human since mammals do not have the corresponding receptors [1-5]. Thus, they have been widely used in genetically modified (GM) crops such as cotton, corn, and soybean to control target pests [6-8]. However, the widespread use of Cry1A toxins has also raised safety fears about possible risks to human health and the ecological environment [9-11]. It has been reported that trace of Cry1Ab have been

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detected in the blood samples from non-pregnant women, mothers, and fetuses [12]. Due to the

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increasing concern about potential risks, it is necessary to develop a detection method that is reliable,

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easy to use and rapid to apply.

Currently, polymerase chain reaction (PCR), high performance liquid chromatography (HPLC),

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and western blot (WB) have been widely used in the detection of Cry toxins in agricultural samples

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[13,14]. However, these methods require expensive instruments, lengthy procedures and experienced technicians [15,16]. Therefore, they are not universally applicable, and a better method is in dire need

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for high-throughput detections and screening of Cry toxins and GM agricultural products. As an alternative, immunoassay methods are simple, rapid and cost-effective which have become the

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preferred alternative methods for routine detections of Cry toxins in agricultural products. At present, the advancement of antibody engineering, such as the recombinant antibody, provides a promising option besides the traditional monoclonal antibody and polyclonal antibody. Single-chain variable fragment (scFv) antibody is composed of the variable regions of heavy (VH) and light chain (VL) of the complete antibody in series with a flexible junction peptide (Linker, 15-25 amino acid residues). They maintain the specific binding activity with the antigens and can be further modified by genetic engineering technology to improve the biological activity [17,18]. Homology modeling can be used to obtain the three-dimensional structure (3D) of Cry toxins and the scFv antibody, followed by the molecular docking to explore the key amino acid binding sites of Cry toxins and the scFv antibody. By combining the two techniques with genetic engineering, it guides the targeted modification of the scFv antibody. Subsequently, the key amino acids were replaced by the other 19 amino acids, and multiple mutants can be obtained in a short time by using the site-saturated 3

Journal Pre-proof mutation technology. Previous report indicated that a mutant with a 20-fold higher affinity than parent was obtained through the mutant library screening, which was established by using sequential error-prone PCR to randomly mutate the scFv antibody [19]. In this study, the DNA of hybridoma cell 5B5 was used as template to amplify the VH and VL gene fragments with universal primers to construct the scFv gene fragment. Then the key amino acid binding sites of scFv to three Cry1A toxins were analyzed using homology modeling and followed by molecular docking. Subsequently, site-saturation mutations of the key amino acids were achieved by

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utilizing the overlap extension polymerase chain reaction (SOE-PCR), and a saturation mutagenesis scFv library was constructed to screen the high-affinity antibodies of the three Cry1A toxins. This study

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immunoassay techniques for the three Cry1A toxins.

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is intended to provide experimental materials and theoretical foundations for establishing generic

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2. Materials and methods

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2.1. Reagents and Materials

Anti-Cry1A mouse hybridoma cell 5B5 and Anti-Cry1 rabbit polyclonal antibodies (PAb) were

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prepared in our laboratory; Cry1 toxins (Youlong Bio. Co., Ltd., China); SuperScriptTM III reverse transcriptase kit and Trizol total RNA isolation kit (Invitrogen, USA); Primers (Shanghai Sangon Bio.

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Co., Ltd., China); AxyPrepTM DNA gel extraction kit, AxyPrepTM PCR clean-up kit and AxyPrepTM midi plasmid kit (Axygen Scientific, Inc., USA); T4 DNA ligase and Restriction endonucleases NcoI, NotI (NEB, China); PMD19-T vector cloning kit (TaKaRa, China); Loading buffer, Goldview, Pfu DNA polymerase, Taq mastermix, DNA marker, E. coli Trans1-T1 competent cells and HRP-anti-his antibody (Beijing TransGen Bio., China); pIT2 expression vector, E. coli TG1 and HB2151 (Tomlinson Library, USA); HiTrap protein G HP column (GE, USA); M13K07 (MRC, England); Isopropyl-β-d-thiogalactoside (IPTG) (Merck, USA); Anti-M13 antibody (HRP) (Sino Biological Inc., China); Anti-mouse antibody (HRP) and Anti-rabbit antibody (HRP) (KPL Inc., MD); All other reagents and chemicals used were of analytical grade or better. 2.2. Cloning of scFv gene from hybridoma and construction of pIT2-scFv expression vector The extraction of total RNA from the hybridoma cells 5B5 were implemented following the instruction of the Trizol total RNA isolation kit. First-strand complementary DNA (cDNA) was reverse 4

Journal Pre-proof transcription synthesized from mRNA utilizing the SuperScriptTM III reverse transcriptase kit. The VH and VL genes of the antibody from cDNA were amplified through PCR. The primer sets for VH and VL gene amplification were shown in Table 1. Subsequently, after purified by DNA gel extraction kit, VH and VL genes were cloned into pMD19-T vector separately, and then transformed into E. coli Trans1-T1 competent cells. The transformants were spread on TYE-AG plates, which were supplemented with ampicillin (100 μg mL−1) and glucose (1%). After an overnight inverted incubation at 37 °C, individual clones were selected from the plates randomly and confirmed by PCR and DNA sequencing (Sangon Bio. Co.,

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Ltd., China) using the universal primer set of pMD19-T vector (Table 2). The positive clones were

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amplified and the plasmids were extracted by using the plasmid mini extraction kit.

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According to the sequencing results of VH and VL genes (Table 3), PCR primer sets were designed (Table 4), and scFv-5B5 was constructed by splicing the VH and VL genes using a flexible linker. First,

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primer sets that are covering restriction enzyme sites and part of the linker (VH-NcoI-F, VH-Linker-R,

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and VL-Linker-F, VL-NotI-R) were used to amplify NcoI-VH-Linker and Linker-VL-NotI genes, respectively. Second, the NcoI-VH-Linker and Linker-VL-NotI genes were added to the PCR system in

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equal quantity, and then the scFv gene was assembled through the overlapping gene of the linker in VH and VL by SOE-PCR using the primer set VH-NcoI-F and VL-NotI-R. After further purification, the

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scFv genes were cloned into pMD19-T vectors and then introduced into E. coli Trans1-T1 competent cells. Positive scFv-5B5 plasmids were extracted and stored at -20 ℃. Afterward, equal quantity of scFv-5B5 plasmids and pIT2 plasmids were digested with NcoI and NotI restriction enzymes, respectively. The digested products were identified by 1% agarose gel electrophoresis and then purified using DNA gel extraction kit. Subsequently, the purified enzyme-digested products of scFv-5B5 and pIT2 plasmids were ligated using T4 DNA ligase to construct the pIT2-scFv-5B5 recombinant vector. Then, pIT2-scFv-5B5 recombinant vectors (10 µL) were introduced into E. coli HB2151 chemical competent cells and incubated with shaking at 200 rpm for 1 h at 37 °C. Subsequently, 100 μL transformation mixture was pipetted and spread on the TYE-AG plate, which was supplemented with ampicillin (100 μg mL−1) and glucose (1%, w/v). After the selection on TYE-AG plate overnight at 37 ℃, one positive clone was selected randomly from the plate and identified by the colony PCR, restriction enzyme analysis (NcoI and NotI), and sequencing by 5

Journal Pre-proof Sangon biotechnology co., LTD (Shanghai, China). The universal primer set LMB3 and pHENseq of pIT2 for PCR and sequencing were shown in Table 2. 2.3. Homology modeling and molecular docking The 3D structures of Cry1Aa, Cry1Ab, and Cry1Ac have obtained and the 3D structure of scFv-5B5 was constructed as described before [20]. Then, molecular docking of scFv-5B5 with three Cry1A toxins was proceeded using the ZDOCK server website (http://zdock.umassmed.edu/), and the structure of complexes was obtained at the same time. The 3D structure was analyzed using

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SWISS-Pdb Viewer (version 4.01) software. Subsequently, the common key amino acid binding sites

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of scFv-5B5 with three Cry1A toxins were further predicted using four hot-point forecast websites (http://robetta.bakerlab.org/alascansubmit.jsp;

http://prism.ccbb.ku.edu.tr/hotpoint/;

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https://mitchell-lab.biochem.wisc.edu/KFC_Server/index.php; http://structure.pitt.edu/anchor/).

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2.4. Construction of site-directed saturation mutagenesis library

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Specific primers were designed according to the gene sequence of scFv-5B5 and site-saturation mutagenesis were performed on the key amino acid sites by SOE-PCR. The PCR products were

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identified by 1% agarose gel electrophoresis and purified using DNA gel extraction kit. Subsequently, the purified DNA gene fragments were digested by restriction enzyme NcoI and NotI and then ligated

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with pIT2 phagemid vectors which have been digested by the same restriction enzymes via T4 DNA ligase. The recombinant pIT2 vectors were then transformed into E. coli TG1 competent cells through electroporation at the power of 2.0 kV and 25 μF capacitance. Immediately after electroporation, 950 μL preheated 2×TY medium was added into the electroporation cuvettes. After gentle blending, the mixture was transferred to 37 °C incubator with shaking at 200 rpm for 1 h to resuscitate the bacterium. 100 μL of bacteria solution was pipetted and gradient diluted at 10 times in 2× TY medium and then spread on TYE-AG plates which supplemented with ampicillin (100 μg mL−1) and glucose (1%). After overnight incubation at 37 °C, the individual clones on the plate were counted first to determine the transformation efficiency and the capacity of the mutagenesis library. Then, five individual clones were selected from the plates randomly for PCR identification to determine the correct insertion. On the other hand, a 5-minute 3000×g centrifuge process was applied to the rest cell cultures, followed by spreading the bacteria precipitate on a TYE-AG plate which supplemented with ampicillin (100 μg 6

Journal Pre-proof mL−1) and glucose (1%). After overnight incubation at 37 °C, 2 mL of 2× TY medium was added to the lawn plate and all the content was then washed down and transferred to a 1.5 mL centrifuge tube. After mixed with glycerol (15%), the bacteria solution was stored at -80 °C, which was called primary site-saturation mutagenesis library. 2.5. Phage antibodies amplification and precipitation The phage antibody amplification and precipitation were performed according to the method described before [20]. Briefly, 1 mL of site-saturation mutagenesis library was added to 200 mL 2×TY

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medium which containing ampicillin (100 μg mL−1) and glucose (1%), and then incubated at 37 °C for

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2-3 h with shaking at 250 rpm until the OD600 of the bacteria solution reached 0.4. Then, 1×1012 M13K07 helper phage was added to the bacteria solution and incubated at 37 °C water bath for 30 min.

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Subsequently, the infected bacterium cells were collected by centrifugation at 3000×g for 10 min and

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resuspended in 400 mL 2× TY medium that contains ampicillin (100 μg mL−1), kanamycin (50 μg mL−1) and glucose (0.1%), and then incubated at 30 °C overnight with shaking at 200 rpm. The overnight

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culture went through a centrifuge process at 3300×g for 30 min, and 100 mL precooled PEG/NaCl (20% polyethylene glycol 6000 in 2.5 M NaCl, w/v) was added to the supernatant. After the mixture was

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thoroughly mixed, it was incubated in an ice bath for 1.5 hours, and then centrifuged at 3300×g for 30 min to remove PEG/NaCl. 2-3 mL of PBS was added to the precipitated phages to prepare for

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subsequent screening.

2.6. Screening and identification of specific phage antibody Positive-negative screening strategy was adopted to screen the specific scFv antibody and isolate non-specific binders from the site-saturation mutagenesis library by using the mixture of Cry1A toxins as coating antigen. The library panning was implemented according to the method described before [21]. Following the method described by Zhang et al. [22] and Jiao et al. [23], the monoclonal phage ELISA was implemented orderly. The titer of the elution was evaluated as described before [20] and the eluted antibodies were used for the next round of panning. The extraction of the positive recombinant plasmid and the transformation to E. coli HB2151 chemical competent cells, as well as the colony PCR, restriction double-enzyme digestion and sequencing were implemented as described in 2.2 section in this paper. 7

Journal Pre-proof 2.7. Expression and determination of anti-Cry1A scFv-5B5 and the mutant antibody The expression and purification procedures of the anti-Cry1A scFv-5B5 and mutant antibody were performed as described before [21]. Briefly, scFv-5B5 and mutant antibody were cultured in 2×TY-AG culture medium that contains ampicillin (100 mg mL-1) and glucose (1%) and incubated overnight at 37 °C with shaking at 200 rpm. Subsequently, the culture medium of the two bacteria were respectively inoculated in 200 mL 2×TY-AG medium that contains ampicillin (100 mg mL-1) and glucose (0.1%) and then incubated at 37 °C for 2-3 h with shaking at 250 rpm. When the OD600 of the

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bacteria solution reached 0.8, 1 mM IPTG was added and the bacterial culture solution was further incubated at 30 °C for 10 h with shaking at 250 rpm. Subsequently, the bacterial culture solution was

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centrifuged at 10000×g for 30 min, and the precipitated bacterial cells were collected and resuspended

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in 20 mL binding buffer (6 g Na3PO4·12H2O, 0.34 g imidazole, and 29.22 g NaCl dissolved in 1 L deionized water, pH 7.4). Then, sonication (35% power, 3 s/4 s, work/stop) was used to lyse cells for

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40 min to release the internal proteins. The lysate was centrifuged at 12000×g for 20 min and the

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supernatant and precipitate were collected respectively. The proteins from periplasm, inclusion body and whole-cell were detected by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE) with non-induced positive bacteria and pIT2 empty plasmid as blank controls. Inoculum enlargements were performed after confirmed the expression location of scFv-5B5 and mutant antibody.

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The scFv antibodies were purified by HiTrap Protein G HP column and then dialyzed against PBS solution at 4 °C for 48 h and then identified using indirect ELISA, SDS-PAGE, and WB techniques. The indirect ELISA was operated following the method described before [22]. The cross-reactivity of scFv-5B5 and mutant antibody with Cry1B, Cry1C and Cry1F were also detected by indirect ELISA. The affinities of scFv-5B5 and mutant antibody with Cry1Ab were measured through surface plasmon resonance (SPR) using a Biacore X100 system (GE Healthcare) as described before [20]. Briefly, Cry1Ab (25 μg mL−1) were immobilized on the CM5 chip, and then different concentrations (5, 10, 20, 40, 80 μg mL−1) of scFv-5B5 and mutant antibody were injected (30 μL min−1) respectively. Finally, all data were analyzed using Biacore X100 evaluation software 2.0.1 version. 2.8. Development and optimization of DAS-ELISA The development and optimization of double antibody sandwich enzyme-linked immunosorbent 8

Journal Pre-proof assay (DAS-ELISA), as well as the calculation of limit of detection (LOD), limit of quantitation (LOQ), and working range were performed according to the method described before [20]. 3. Results 3.1. Cloning of scFv gene from hybridoma and construction of pIT2-scFv expression vector First, the total RNA of anti-Cry1A mouse hybridoma cells 5B5 was extracted and reverse translated into cDNA successfully. Then, using cDNA as template, VH and VL genes were amplified with mixed primer sets VHF-1-19, VHR-1-4, and VLF-1-1, VLR-1-3 respectively, and then cloned into

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PMD19-T vector, as well as confirmed by PCR with the universal primer set of pMD19-T (RV-M,

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M13-47) (Fig. S1a). Subsequently, the VH-Linker (about 396 bp), and VL-Linker (about 375 bp) were

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amplified with primer sets VH-NcoI-F, VH-Linker-R, and VL-Linker-F, VL-NotI-R respectively, and the scFv gene (about 750 bp) was constructed by splicing the V H-Linker and VL-Linker through SOE-PCR

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VL (about 342 bp) were shown in Table 3.

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with primer set VH-NcoI-F and VL-NotI-R (Fig. S1a). The sequencing results of VH (about 363 bp) and

Subsequently, the scFv-5B5 genes were digested and cloned into pIT2 plasmids. Then the

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recombinant pIT2 plasmids were identified by PCR and restriction double-enzyme digestion (Fig. S1b). The sequencing result showed that the scFv-5B5 gene was successfully constructed and inserted into

Fig. S1c.

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the pIT2 vector's reading frame, and the amino acid sequence of scFv-5B5 was deduced and shown in

3.2. Homology modeling and molecular docking The 3D structure of scFv-5B5 was constructed successfully using the homology modeling website. Then, based on the 3D structures of scFv-5B5 and three Cry1A toxins, the Cry1A-scFv complexes were obtained using molecular docking. The amino acid sequences and 3D structures of Cry1Aa, Cry1Ab, and Cry1Ac are highly homologous and the binding hot sites with scFv-5B5 were identical. In this case, represented by Cry1Ab, the results were shown in Table 5 and Fig. 1. The molecular docking results showed that the binding hot sites were located in domain I on Cry1A toxins, and VH complementarity-determining region (CDR1 and CDR3) and VL framework region (FR3) on scFv-5B5. The hot point prediction results showed that 35th, 36th, 104th, 105th, and 196th amino acid residues were predicted as key amino acid binding sites on scFv-5B5 by more than two hotspot prediction websites 9

Journal Pre-proof (Table 5) which were consistent with the molecular docking results. There are five hydrogen bonds between the five key amino acid binding sites and the Cry1Ab toxin (Fig. 1). 3.3. Construction of site-directed saturation mutagenesis library Specific primer sets (Table 6) were used to carry out site-directed mutations on 35th, 36th, 104th, 105th, and 196th key amino acid sites by SOE-PCR according to the amplification steps in Table 7. The results showed that the mutant fragments 1, 2, 3 and 4 were amplified successfully. The scFv-5B5 mutant genes were constructed by splicing fragments 1, 2, 3, and 4 (Fig. S2). Then, scFv-5B5 mutant

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genes were cloned into the pIT2 phagemid vectors and transformed into E. coli TG1 and a site-directed

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saturation mutagenesis library with a capacity of 1.35×10 5 CFU/mL was constructed successfully. Five positive clones were selected randomly from the plates for sequencing purpose, and the results showed

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that no identical sequences were obtained (Fig. 2), which indicated the correct insertion of the scFv

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five target sites was performed successfully.

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genes and the excellent gene diversity of the library, as well as the fact that the synergic mutation in the

3.4. Screening and identification of specific phage antibody

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After four rounds of panning, one positive phage-scFv mutant clone scFv-2G12 (absorptive value of positive clone/absorptive value of negative clone > 2.1) was selected from the fourth round of plates.

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As demonstrated in Fig. 3, the levels of binding activity of scFv-2G12 to Cry1Aa, Cry1Ab, and Cry1Ac toxins were 1.68-fold, 1.40-fold, and 2.06-fold greater than that of maternal antibody scFv-5B5, respectively. Then scFv-2G12 was identified by colony PCR and restriction double-enzyme digestion and the result demonstrated that the insertion of scFv-2G12 was correct (about 950 bp) (Fig. S3). Meanwhile, sequencing results showed that scFv-2G12 has two amino acid sites mutation at 36th and 196th, which modified from phenylalanine36 and serine196 in scFv-5B5 to tyrosine36 and tyrosine196 (Fig. 4a). 3.5. Expression and determination of anti-Cry1A scFv-5B5 and scFv-2G12 The soluble antibody fragments scFv-5B5 and scFv-2G12 were successfully expressed in E. coli HB2151 after cultured at 30 °C for 10 h with 1 mM IPTG. Then, the soluble antibody fragments scFv-5B5 and scFv-2G12 were purified by HiTrap Protein G HP column and identified by SDS-PAGE and WB. According to the results, only a single protein band approximately 27 kD was eluted down 10

Journal Pre-proof from the column for scFv-5B5 and scFv-2G12 (Fig. 4b). Furthermore, WB results were consistent with SDS-PAGE (Fig. 4c). The indirect ELISA results indicated that both scFv-2G12 and scFv-5B5 showed generic binding activities to the three Cry1A toxins and no cross-reactivity were observed with Cry1B, Cry1C, and Cry1F. The binding activities of scFv-2G12 to the three Cry1A toxins were generally higher than that of scFv-5B5 (Fig. 4d). The results of the molecular docking of scFv-2G12 with Cry1Ab showed that the number of hydrogen bonds increased (Fig. 5). Then, the purified scFv-5B5 and scFv-2G12

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antibodies were used to establish the DAS-ELISA method.

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The affinities of scFv-5B5 and scFv-2G12 with Cry1Ab toxin were measured and the results indicated that mutant antibody scFv-2G12 presented higher affinity with Cry1Ab toxin than the

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the results were consistent with the indirect ELISA.

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maternal antibody scFv-5B5, which were 9.819×10-9 M and 2.025×10-8 M, respectively (Fig. 6). And

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3.6. Development and optimization of DAS-ELISA

The optimal concentrations of capture antibody (anti-Cry1A scFv-5B5, scFv-2G12) and detection

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antibody (anti-Cry1 rabbit Pab) in DAS-ELISA were calculated to be 3.5 and 0.2 μg·mL−1, respectively. Then, based on the optimized antibody concentrations, standard curves for three Cry1A toxins were

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constructed (Fig. 7). The results presented a linear relationship between the absorbance values at OD450 and the concentrations of three Cry1A toxins up to 32 μg·mL−1. As shown in Table 8, the LOD values for scFv-2G12 and scFv-5B5 were 4.6-9.2 and 12.4-22.0 ng mL−1, respectively, and LOQ values for scFv-2G12 and scFv-5B5 were 11.1-17.1 and 23.6-39.7 ng mL−1, respectively. ScFv-2G12 against Cry1Aa, Cry1Ab, and Cry1Ac had lower LOD and LOQ values than those of the maternal antibody scFv-5B5. In addition, the quantitation ranges for scFv-2G12 were broader than scFv-5B5 (Table 8). 3.7. Comparison with some published results At present, many researches have produced anti-Cry1 scFv antibodies and established the corresponding ELISA methods to detect one or more Cry1 toxins. We compared the results of this study with the published results, which revealed that the established DAS-ELISA method based on scFv-2G12 can specific detect three Cry1A toxins simultaneously with low LOD values, without interference from other Cry1 toxins (Table 9). 11

Journal Pre-proof 4. Discussion ScFv is a kind of fusion protein that is assembled by the VH and VL of the complete antibody through a flexible linker peptide containing about 15-25 amino acids, and retains the specific binding activity of the complete antibody. ScFv can be constructed by cloning the VH and VL genes derived from hybridoma cells and produced on a large scale using prokaryotic expression systems [26-28]. The scFv has characteristics such as small molecular weight, rapid preparation, low cost and mass production without immunization. What's more, the relationship between the structure and function of

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scFv can be studied at the molecular level, and the affinity of scFv with antigen can be further improved through gene mutation. Therefore, scFv antibody is widely used in the detection of Cry

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toxins and pesticide residues in environment or agricultural products [29,30]. The premise of

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constructing a functional scFv is to amplify the variable region of the antibody correctly. In this study, hybridoma cells 5B5 that secrete anti-Cry1A mAb were used as the resource of the antibody variable

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region gene. Compared with the antibody library screening method, scFvs can be obtained more faster

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and more targeted [24,27].

On the other hand, the appropriate expression system and strategy are the key factors to obtain the

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active recombinant scFv antibody. At present, the E.coli expression system has become the most widely used prokaryotic expression system due to its relatively clear genetic background, simple operations

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and the ability to express a large number of exogenous genes [31]. In this study, the scFv gene was successfully cloned into the prokaryotic expression vector pIT2 and expressed in large quantities in E. coli HB2151. The expression products were identified by indirect ELISA and showed that it has the same recognition spectrum as the parent mAb which can recognize three Cry1A toxins. However, scFv has a lower affinity than their parent mAb, which is consistent with many previous studies and therefore limits their further application [32]. In an attempt to solve this problem, the site-directed saturation mutagenesis library was constructed to screen scFv mutant with higher affinities. Phage antibody library technology is developed based on the phage display technology and it is a new gene engineering antibody technology, which was simple and effective for the screening of the target antibody. Based on the unique advantage of phage antibody display technology in unifying "genotype" and "phenotype", once the target antibody was screened out, the corresponding gene sequence was obtained at the same time. 12

Journal Pre-proof With the rapid development of computer techniques, computational simulations such as homologous modeling and molecular docking are widely adopted to predict the key amino acid binding sites in antigens and antibodies, which provides a theoretical basis for the modification of antibodies by genetic engineering technology [33]. Therefore, homology modeling and molecular docking techniques can be used to predict the key sites where antibodies bind to antigens, after which a variety of affinity maturation techniques in vitro were used to modify the antibodies [34,35]. It is more probable to obtain the mutation antibody with an optimal performance by introducing mutations in the hot spots rather than in other areas [36]. In this study, to increase the affinity of scFv-5B5, homologous modeling and

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molecular docking were used to predict the key amino acid binding sites, and the result showed that

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35th, 36th, 104th, 105th, and 196th residues were predicted as the key amino acid binding sites in

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scFv-5B5. There are four binding sites in scFv-5B5 located in the CDR1 and CDR3 of VH and one binding site located in the FR1 of VL. CDRs are common mutation sites in the antibody and can greatly

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influence the activity of antibody [37,38]. However, there are also study suggests that mutations in the

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FR region of the antibody can also affect the binding activity [39]. Site mutations lead to tiny variation in the structure of antibodies and make the complementary domain of antibodies and antigens more

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complete [40]. Studies have shown that multiple-site mutations can introduce positive cooperative effects, leading to conformational changes in proteins, and the mutants is more efficient than single

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site-directed mutation or random mutation [41]. Then, through saturation mutations at the key amino acid sites, a site-directed saturation mutagenesis phage display scFv library (1.35×105 CFU/mL) was constructed successfully. After four rounds of screening, one positive mutant clone scFv-2G12 was obtained and then expressed in E. coli HB2151, as well as validated by ELISA, which showed that compared with scFv-5B5, the recognition abilities of scFv-2G12 with the three Cry1A toxins were significantly improved. Molecular docking of scFv-2G12 with Cry1Ab showed that the number of hydrogen bonds increased. However, the affinities of scFv-2G12 with the three Cry1A toxins were less improved than expected, for which there are several possible explanations. One of them is that the scale of the randomized mutations is limited, and some mutation sites have non-affinity effects or the most critical sites were not mutated. Another reason may be that the library capacity is not large enough to screen out scFv antibodies with higher affinities to the three Cry1A toxins. The screening method is another important factor, and some scFvs with high-affinity may not be enriched by using the mixture of three Cry1A toxins as coating antigen in 13

Journal Pre-proof the process of screening for the aiming was to obtain broad-spectrum scFvs. Nevertheless, the mutant scFv-2G12 is still a decent material to establish the DAS-ELISA for the detection of three Cry1A toxins, as well as providing material for further study. DAS-ELISA is the most commonly used immunoassay method for the detection of macromolecular antigen in scientific research and commercial ELISA kits and it is superior to indirect competitive ELISA in sensitivity and working range [42]. In our study, the LOD and LOQ for the three Cry1A toxins of the established DAS-ELISA based on scFv-2G12 were 4.6-9.2 ng mL-1 and 11.1-17.1 ng mL-1 respectively, which were less sensitive than those of commercial ELISA kits in terms of the

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detection of single toxin [43-45]. This is because the application of genetically engineered antibodies in

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the food safety detection is still in the early stage and there are some deficiencies in the practical

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application. With the development of molecular biology, the performance of genetically engineered antibodies will be continuously developed and improved, which is believed to revolutionize the

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detection methods in food safety [46,47].

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5. Conclusion

In order to obtain high affinity antibody materials for the detection of Cry1A toxins. ScFv

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antibody was amplified from hybridoma cell 5B5 which secreted anti-Cry1A mAb. Subsequently, with the help of homology modeling and molecular docking techniques, the key amino acid binding sites of

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scFv-5B5 with three Cry1A toxins were predicted and site-saturation mutagenesis was performed. Then, a site-saturation mutagenesis scFv library with a capacity of 1.35×105 CFU/mL was constructed successfully. With the mixture of three Cry1A toxins as coating antigen, a high-affinity scFv-5B5 mutant (scFv-2G12) which could detect three Cry1A toxins simultaneously was screened out and used to establish the DAS-ELISA, which showed that the LOD and LOQ values of scFv-2G12 against Cry1Aa, Cry1Ab and Cry1Ac were lower than maternal antibody scFv-5B5. The results indicated that scFv-2G12 was a promising material for the development of high sensitive immunoassay technology for Cry1A toxins.

14

Journal Pre-proof Acknowledgements This work was supported by the Natural Science Foundation of Jiangsu Province (BK20180916); the Special Financial Grant from the China Postdoctoral Science Foundation (2018T110464); the General Financial Grant from the China Postdoctoral Science Foundation (2017M621674 and

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2018M630538); and the National Natural Science Foundation of China (31630061).

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Author Contribution Statement Sa Dong: Conceptualization, Methodology, Writing-Original draft preparation, Funding acquisition. Meijing Gao: Methodology, Funding acquisition. Zongyi Bo: Methodology, Data curation, Validation. Lingjun Guan: Investigation, Data Curation. Xiaodan Hu: Data Curation. Hanxiaoya Zhang: Writing - Reviewing. Beibei Liu: Software. Pan Li: Software. Kangli He:

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Investigation. Xianjin Liu: Formal analysis, Funding acquisition. Cunzheng Zhang: Writing -

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Reviewing and Editing.

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Journal Pre-proof Figure captions:

Fig. 1. The results of the molecular docking of scFv-5B5 with Cry1Ab toxin. Fig. 2. Sequence alignment between mutations and maternal scFv-5B5. Fig. 3. Monoclonal phage ELISA against three Cry1A toxins. The positive phage infected E. coli TG1 culture supernatant containing approximately 1 × 108 phage particles per milliliter was analyzed by monoclonal phage ELISA. Absorbance values are arithmetic means of triplicate measurements and means separated with the Student-NewmanKeuls test (P < 0.05). Bars with the same background images followed by the same letter did not

of

differ at the 5% significance level. Error bar indicates the standard deviation.

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Fig. 4. The deduced amino acid sequence of scFv-2G12, SDS-PAGE, and WB analysis of the purification of

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scFv-5B5 and scFv-2G12, as well as the binding activities of scFv-5B5 and scFv-2G12 to six Cry1 toxins. (a) Deduced amino acid sequence of scFv-2G12 according to the nucleotide sequence. (b) Lane M: protein marker,

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SDS-PAGE analysis of the purified soluble scFv-5B5 protein (left to right: 8, 0.6 and 1.2 μg) and scFv-2G12 protein

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(left to right: 9.5, 1.8 and 1.4 μg). (c) WB analysis of the purified soluble scFv-5B5 and scFv-2G12 protein. (d) -1

Binding activities of the purified soluble scFv-5B5 and scFv-2G12 protein (200 μg mL ) to six Bt toxins (coating -1

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concentration of 4 μg mL , HRP/anti-E Tag mouse monoclonal antibody were 1:4000 diluted in MPBS) determined by indirect ELISA. Absorbance values are arithmetic means of triplicate measurements and means separated with

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the Student-Newman-Keuls test (P < 0.05). Bars followed by the same letter did not differ at the 5% significance level. Error bar indicates the standard deviation.

Fig. 5. The results of the molecular docking of scFv-2G12 with Cry1Ab toxin. Fig. 6. Sensograms and binding kinetic parameters of Cry1Ab toxin with scFv-5B5 and scFv-2G12. Fig. 7. Construction of standard curves of scFv-5B5 and scFv-2G12 for three Cry1A toxins based on scFv (3.5 μg -1

-1

mL ) and anti-Cry1 rabbit PAbs (0.2 μg mL ) using DAS-ELISA.

22

Journal Pre-proof Table 1 Primer sequences used for variable fragments genes of VH and VL amplification. Primer name

Primer sequence（5’-3’） ATGGCCCTCGAGGTRMAGCTTCAGGAGTC

VHF-2

ATGGCCCTCGAGGTBCAGCTBCAGCAGTC

VHF-3

ATGGCCCTCGAGGTGCAGCTGAAGSASTC

VHF-4

ATGGCCCTCGAGGTCCARCTGCAACARTC

VHF-5

ATGGCCCTCGAGGTYCAGCTBCAGCARTC

VHF-6

ATGGCCCTCGAGGTYCARCTGCAGCAGTC

VHF-7

ATGGCCCTCGAGGTCCACGTGAAGCAGTC

VHF-8

ATGGCCCTCGAGGTGAASSTGGTGGAATC

VHF-9

ATGGCCCTCGAGGTGAWGYTGGTGGAGTC

VHF-10

ATGGCCCTCGAGGTGCAGSKGGTGGAGTC

VHF-11

ATGGCCCTCGAGGTGCAMCTGGTGGAGTC

VHF-12

ATGGCCCTCGAGGTGAAGCTGATGGARTC

VHF-13

ATGGCCCTCGAGGTGCARCTTGTTGAGTC

VHF-14

ATGGCCCTCGAGGTRAAGCTTCTCGAGTC

VHF-15

ATGGCCCTCGAGGTGAARSTTGAGGAGTC

VHF-16

ATGGCCCTCGAGGTTACTCTRAAAGWGTSTG

VHF-17

ATGGCCCTCGAGGTCCAACTVCAGCARCC

VHF-18

ATGGCCCTCGAGGTGAACTTGGAAGTGTC

VHF-19

ATGGCCCTCGAGGTGAAGGTCATCGAGTC

VHR-1

TGAGGAGACGGTGACCGTGGT

VHR-2

TGAGGAGACTGTGAGAGTGGT

VHR-3

TGCAGAGACAGTGACCAGAGT

VHR-3

TGAGGAGACGGTGACTGAGGT

Primers for VL

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VHF-1

GAYATCCAGCTGACTCAGCC

VLF-2

GAYATTGTTCTCWCCCAGTC

VLF-4 VLF-5 VLF-6 VLF-7 VLF-8

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VLF-1 VLF-3

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Primers for VH

GAYATTGTGMTMACTCAGTC GAYATTGTGYTRACACAGTC GAYATTGTRATGACMCAGTC GAYATTMAGATRAMCCAGTC GAYATTCAGATGAYDCAGTC GAYATYCAGATGACACAGAC

VLF-9

GAYATTGTTCTCAWCCAGTC

VLF-10

GAYATTGWGCTSACCCAATC

VLF-11

GAYATTSTRATGACCCARTC

VLF-12

GAYATTKTGATGACCCARAC

VLF-13

GAYATTGTGATGACBCAGKC

VLF-14

GAYATTGTGATAACYCAGGA

VLF-15

GAYATTGTGATGACCCAGWT

VLF-16

GAYATTGTGATGACACAACC

VLF-17

GAYATTTTGCTGACTCAGTC

VLR-1

AGATGGTGCAGCCACAGTTCGTTTKATTTCCAGYTTGGTCCC

VLR-2

AGATGGTGCAGCCACAGTTCGTTTTATTTCCAACTTTGTCCC

VLR-3

AGATGGTGCAGCCACAGTTCGTTTCAGCTCCAGCTTGGTCCC

Note：Y=C/T；B=C/G；W=A/T； S=G/C；M=A/C；R=A/G；K=G/T；D= A/G /T；V= A/C/G 23

Journal Pre-proof Table 2 Universal primer sequences of pMD19-T and pIT2 vectors. Vector name

pMD19-T

Primer name

Primer sequence（5’-3’）

RV-M

GAGCGGATAACAATTTCACACAGG

M13-47

CGCCAGGGTTTTCCCAGTCACGAC

LMB3

CAGGAAACAGCTATGAC

pHENseq

CTATGCGGCCCCATTCA

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pIT2

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Journal Pre-proof Table 3 The sequencing results of VH and VL. Name

Nucleotide sequence

VH

GAGGTGAAGTTGGTGGAGTCAGGACCCAGCCTAGTGCAGCCCTCACAGAGCCTGTCCACAACTTGCACAG TCTCTGGTTTCTCATCAACTAACTTTGGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGG CTGGGAGTGATATGGAGAGGTGGAAACACAGACTACAATGCAGCTTTCATGTCCAGACTGAGCATCACCA GGGACAACTCCAAGAGACAAATTTTCTTTAAAATGGACAGTCTGCAAGCTGATGACACTGCCATATACTACT GTGCCAAACCCCTTATCTACTATGGTAACTACGGTTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCG TCTCCTCA GATATTCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGG

of

GCAAGTCAGGACATTAGAAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTGAACTCCTGATC TACTACACATCAAGGTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACACATTATTCT

ro

CTCGCCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACCTTTGCCAACAGGGTCATGCGCTTCCGTAC

na

lP

re

-p

ACGTTCGGAGGGGGGACCAAGCTGGAGCTGAAACGAACTGTGGCTGCACCATCT

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VL

25

Journal Pre-proof Table 4 Primer sets used for full length scFv gene amplification by SOE-PCR. Primer name VH-NcoI-F

Primer sequence（5’-3’） CATGCCATGGGCGAGGTGAAGTTGGTGGAGTC GCCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGGTGACCGTGG

VL-Linker-F

TCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGATATTCAGATGACACAGAC

VL-NotI-R

ATAAGAATGCGGCCGCAGATGGTGCAGCCACAGTTC

Jo ur

na

lP

re

-p

ro

of

VH-Linker-R

26

Journal Pre-proof Table 5 Hot-point prediction results of the scFv-5B5. Residue ASN

35

Robetta

Drugscore-PPI

√

√

a

√ √

√

√

104

√

√

√

105

√

√

√

196

√

TYR SER

√

√

na

lP

re

-p

ro

of

The amino acid residue was predicted as hot spot by the corresponding hot-point websites.

Jo ur

a

KFC2

36

PHE ILE

Anchor database

27

Journal Pre-proof Table 6 Specific primers used for saturation mutagenesis at target amino acid sites of 35th, 36th, 104th, 105th and 196th in scFv-5B5. Primer name

Primer sequence（5’-3’） CGGCGATGGCCATGGGCGAGGTGAAGTTGGT

1F1

GTCTCTGGTTTCTCATCAACTNNKNNKGGTGTACACTGGGTTCGCCAGTC

1F2

TACTACTGTGCCAAACCCCTTNNKNNKTATGGTAACTACGGTTACTTCG

1F3

TACTACACATCAAGGTTACACNNKGGAGTCCCATCAAGGTTCAGTG

1R

CTCGAGTGCGGCCGCAGATGGTGCAGCCACAGTTCGT

1R1

AGTTGATGAGAAACCAGAGAC

1R2

AAGGGGTTTGGCACAGTAGTATATG

1R3

GTGTAACCTTGATGTGTAGTA

Jo ur

na

lP

re

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ro

of

1F

28

Journal Pre-proof Table 7 The PCR amplification steps of scFv-5B5 mutant.

Template name

Primer –F/Primer -R

Product length (bp)

1

scFv-5B5

1F /1R1

107

2

scFv-5B5

1F1/1R2

228

3

scFv-5B5

1F2/1R3

297

4

scFv-5B5

1F3 /1R

213

scFv-5B5 mutant

1+2+3+4

1F3/1R

782

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Fragment name

29

Journal Pre-proof Table 8 Theoretical values of LOD, LOQ and quantitation range for three Cry1A toxins of scFv-5B5 and scFv-2G12. −1

−1

−1

Toxins

LOD (ng mL )

LOQ (ng mL )

Quantitation range (ng mL )

scFv-5B5

Cry1Aa

13.9

28.5

29–250

Cry1Ab

12.4

23.6

24–250

Cry1Ac

22

39.7

40–250

Cry1Aa

8.5

17.1

18–250

Cry1Ab

9.2

15.3

16–250

Cry1Ac

4.6

11.1

12–250

ro -p re lP na Jo ur

scFv-2G12

of

Antibodies

30

Journal Pre-proof Table 9 Comparison of some published results in recent years of anti-Cry1 scFv antibodies with our research. Year

a

Antibody

-1

Experiment method

LOD (ng mL ) Cry1Aa

Cry1Ab

Cry1Ac

scFv

DAS-ELISA

8.5

9.2

4.6

6.41

11.07

3.79

[21]

2018

scFv

DAS-ELISA

[22]

2014

scFv

DAS-ELISA

[24]

2012

scFv

Ic-ELISA

[25]

2012

scFv

Ic-ELISA

Cry1B

Cry1C

Cry1F

4.56

5.00

3.14

8

b

50

The data were from scFv-2G12 obtained in this study

b

Indirect competitive ELISA

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Literature no.

31

23

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7