Construction of an Immunized Rabbit Phage Display Library for

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Construction of an immunized rabbit phage display library for selecting highactivity of against Bacillus thuringiensis Cry1F toxin single-chain antibodies Chongxin Xu, Cunzheng Zhang, Jianfeng Zhong, Hui Hu, Shimin Luo, Xiaoqin Liu, Xiao Zhang, Yuan Liu, and Xianjin Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01985 • Publication Date (Web): 16 Jun 2017 Downloaded from http://pubs.acs.org on June 19, 2017

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Journal of Agricultural and Food Chemistry

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Construction of an immunized rabbit phage display library for selecting high-activity

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of against Bacillus thuringiensis Cry1F toxin single-chain antibodies

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Chongxin Xua, Cunzheng Zhanga, Jianfeng Zhonga, Hui Hub, Shimin Luob, Xiaoqin Liub, Xiao Zhanga,

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Yuan Liua, Xianjin Liua*

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a

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Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural Sciences, Nanjing 210014,

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China

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b

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

Huaihua Vocational and Technical College, Huaihua 418007, China

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*Corresponding author at: Institute of Food Safety and Nutrition, Jiangsu Academy of Agricultural

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Sciences, Nanjing 210014, China.

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E-mail address: [email protected](Xianjin Liu).

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ACS Paragon Plus Environment


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Abstract: In the present study, a Cry1F-immunized rabbit phage display library (6.96×108) was

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constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (scFv) by bio-panning.

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Total 16 positive monoclonal phage scFvs were obtained after 4 rounds panning which identified by

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enzyme-linked immunoassay (ELISA), PCR and DNA sequencing. The most positive phage scFv

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(named RF4) was expressed in E. coli.HB2151 and the purified soluble protein approximate 30 kDa

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with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). An indirect-competitive

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ELISA (IC-ELISA) was established base on purified soluble RF4-scFv for Cry1F toxin and indicated

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the 50 % inhibition of control (IC50) was 11.56 ng/mL, the detection limit (IC10) was 0.18 ng/mL, and

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showed a weakly cross-reactivity (CR) for Cry1Ab (2.8 %), Cry1Ac (1.3 %), Cry1B, Cry1C, Cry1Ie

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and Cry2A (less than 0.1 %). It had a good accuracy, stability and repeatability that Cry1F toxin spiked

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in rice, wheat, corn and soil samples by IC-ELISA, the recoveries were in the range of 80.2 %~99.6 %

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and the coefficient of variation (CVs) were among 2.5 %~10.0 %. These results showed the IC-ELISA

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based on scFv from the immunized rabbit phage display library was promising for specific-detecting

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Cry1F toxin in agro-products and environmental samples.

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Keywords: Phage display library; Rabbit scFv; Bacillus thuringiensis; Cry1F toxin; ELISA

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Introduction

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Cry1F toxin is a type of Bt Cry toxins that produced by Bacillus thuringiensis with the process of

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spore formation1, 2. It has a role for killing many kinds of agricultural pests, especially for lepidoptera

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insects, and has been used for genetically modified crops (GMCs) in rice, wheat, corn, cotton, etc. so

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far3-6. However, the problem of GMCs biosafety risk has been a focus between approvers and

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opponents in the international community7, 8. In recent years, some research evidence suggests that the

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GMCs maybe exert the risk of accelerating the frequency of insect resistance evolution, breaking the

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original soil environment balance of biological community, facilitating the gene drift of cross-species

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and threating the mammals of normal immune system, etc.9-12. Therefore, it is important to detect

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GMCs and their products for agriculture and environment13-15.

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At present, phage display library combines the antigen-specific phage displayed-antibody

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bio-panning technology is very popular for rapid preparation of genetically engineered artificial

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antibodies in biomedicine and immunoassay fields16, 17. This technique was first described by Dr. Smith

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in 1985, which cloned artificial antibody genes into a characteristic reformed phagemid vector and the

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antibody proteins were displayed on the surface of filamentous phage capsid proteins by

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co-expression18. Such fusion can be obtained not only from the antibody, but also from its gene, which

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coats antigen in plastic cell culture flask to capture the antigen-specific binding phage

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displayed-antibody by bio-panning from the library19. Phage displayed-antibody unlike traditional

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polyclonal antibody and monoclonal antibody preparation of technology, it can rapidly and effectively

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avoid the cumbersome process without immunization of animals. At the same time, the obtained

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antibody gene would be used for further clonal expression in different vectors or affinity mature

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evolution, conveniently20-23. Qiu et al.24 have employed a naive camel phage display nanobody 3 / 25



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library to successfully select anti-deoxynivalenol (DON) nanobody. Pansri et al.25 constructed a native

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human phage scFv library and used for selection of anti-snake venom and anti-Aflatoxin B1 scFvs

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successfully. In previous studies, our research team had also obtained series of anti-Bt Cry toxins scFvs

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from a commercialization of naive human semi-synthetic phage display libraries (Tomlinson I + J) and

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established the ELISA for corresponding of Bt Cry toxins26-28. However, it is hard to obtain the high

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affinity of antigen-specific phage antibody compared with the traditional polyclonal antibody and

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monoclonal antibody that from the naive phage display library without antigen-immunization instead

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by bio-panning directly29-31.

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In order to rapidly and effectively obtain the high affinity of Cry1F-specific phage scFvs for

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analysis of Bacillus thuringiensis Cry1F toxin residual in agro-products and environmental samples in

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the present study, first of all, constructing a Cry1F-immunized rabbit phage display library for

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screening the anti-Cry1F phage displayed scFvs, then expressing and purifying the soluble scFv

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proteins in E. coli.HB2151, finally, establishing an IC-ELISA based on purified scFvs for detecting

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Cry1F toxin and evaluating its practicality and applicability by spiked in rice, wheat, corn, soil, etc.

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Materials and methods

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Bt Cry toxins standards were all purchased from You Long Bio.Co.Ltd (Shanghai, China). New

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Zealand white rabbit used in this experiment were supported by National Research Center of Veterinary

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Biologicals engineering and technology (Nanjing, China) and the experiment protocol was approved by

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the animal experiment ethics committee of authors’ institute. pIT2 phagemid vector (HIS myc tag), E.

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coli.TG1, E. coli.HB2151, KM13 helper phage were obtained from MRC HGMP Resource Centre

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(Cambridge, England). Goat Anti-Rabbit IgG-[HRP], Anti-M13-[HRP] monoclonal antibody, Anti-HIS

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tag [HRP] monoclonal antibody were purchased from GenScript Bio.Co.Ltd (Nanjing, China). Cell 4 / 25


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culture flask, 96-well plates were purchased from Corning (Beijing, China). Primers used in

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experiments (Table 1) all synthesize from Sangon Bio.Co.Ltd. (Shanghai, China). Kits for total RNA

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isolation system, reverse transcription system and mRNA isolation system, PCR Mix system, NotI and

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NcoI restriction endonucleases, T4 DNA ligase were purchased from Promega. Other reagents were all

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purchased from GE Healthcare (Beijing, China).

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Immunization of rabbit. A healthy New Zealand white rabbit was immunized by four times

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injections of Cry1F toxin mixed with Freund’s adjuvant, to stimulate B-cells expressing anti-Cry1F

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toxin polyclonal antibodies. In briefly, in the first immunization, after collecting blood from ear vein

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(negative serum), 1 mL Cry1F toxin [dissolved in CBS buffer (1 mg/mL)], mixed with 1 mL Freund’s

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complete adjuvant when completing emulsification for subcutaneous injection. While the second and

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third immunizations were injected every two weeks, respectively, the methods were the same as the

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first time, with Freund’s complete adjuvant replaced by Freund’s incomplete adjuvant. A week later,

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collecting blood after the third immunization and testing the serum titer, when reached to 105 for

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enhancing immunization (the fourth) with 1 mL Cry1F toxin (1 mg/mL) immediately. Another week

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later, collecting spleen tissue for constructing the Cry1F-immunized rabbit scFv library.

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Construction of phage display scFv library. Total RNA was extracted from the spleen tissue of

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Cry1F-immunized rabbit using Trizol Reagent (Invitrogen, USA) in accordance with the product

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operation manual, its purity and concentration were calculated by UV absorbance at OD260/OD28031.

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The first strand cDNA was generated by using PrimeScript™ II 1st Strand cDNA Synthesis Kit

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[TaKaRa (Dalian), China] according to the product operation manual. The primers used for PCR

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amplification of constructing phage display scFv library were designed as the Table 1 and all dissolved

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in ddH2O with a concentration of quantified for µM. All the PCR products were purified by 5 / 25



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GenElute™ PCR Clean-Up Kit or GenElute™ Gel Extraction Kit

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ddH2O with a concentration of quantified for µM, then used for follow PCR reaction of templates.

(Sigma, USA), finally dissolved in

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Firstly, VL (light chain variable region) and VH (heavy chain variable region) were amplified by

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PCR respectively. VL PCR reaction system: 20 µL system contains 1 µL cDNA, 1 µL upstream primer

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(RLF1、RLF2、RLF3、RLF4 mixed with 1:1:1:1), 1 µL downstream primer (RLB1, RLB2, RLB3,

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RLB4 mixed with 1:1:1:1). VH PCR reaction system: 20 µL system containing 1 µL cDNA, 1 µL

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upstream primer (RHF1, RHF2, RHF3, RHF4 mixed with 1:1:1:1), 1 µL downstream primer (RHB).

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All their PCR reactions condition were: 95 ℃ 10 min, then 94 ℃ 1 min, 56 ℃ 1 min, 72 ℃ 1 min

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for 30 cycles, and final extension at 72 ℃ for 10 min. Secondly, VL and VH were added Linker gene

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turn into VL-Linker and Linker-VH by PCR respectively. VL-Linker PCR reaction system: 50 µL system

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contains 1 µL VL PCR products, 1 µL upstream primer (RLF1, RLF2, RLF3, RLF4 mixed with 1:1:1:1),

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1 µL downstream primer (VL-Linker). Linker-VH PCR reaction system: 20 µL system contains 1 µL VH

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PCR products, 1 µL upstream primer (Linker-VH), 1 µL downstream primer (RHB). All their PCR

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reaction conditions were: 95 ℃ 10 min, then 95 ℃ 1 min, 57 ℃ 1 min, 72 ℃ 2.5 min for 30

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cycles, and final extension at 72 ℃ for 10 min. Then, splicing the VL-Linker and Linker-VH sequences

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by overlap extension PCR (SOE-PCR) to a whole scFv. SOE-PCR reaction system: 50 µL system

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contains 1 µL VL-Linker PCR products, 1 µL Linker-VH PCR products, 1 µL upstream primer (RLF1、

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RLF2、RLF3、RLF4 mixed with 1:1:1:1), 1 µL downstream primer (RHB). SOE-PCR reaction

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condition was: 95 ℃ 10 min, then 94 ℃ 1.5 min, 58 ℃ 1 min, 72 ℃ 2.5 min for 30 cycles, and

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final extension at 72 ℃ for 10 min. Finally, the scFv PCR products added NotI and NcoI restriction

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sites sequences by PCR amplification using RF-NcoI and RB-NotI primers. PCR reaction system: 50

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µL system contains 0.5 µL scFv PCR products, 1 µL upstream primer (RF-NcoI), 1 µL downstream 6 / 25


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primer (RB-NotI). PCR reaction condition was: 95 ℃ 10 min, then 95 ℃ 2 min, 58 ℃ 1.5 min,

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72 ℃ 1 min for 30 cycles, and final extension at 72 ℃ for 10 min. The purified PCR products of

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scFv with NotI and NcoI restriction sites sequences were double digested by NotI and NcoI at 37 ℃

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for overnight, then the fragment were purified and ligated into pIT2 phagemid vector which have been

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digested by the same restriction endonucleases. The pIT2-scFv recombinant phagemids were

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transformed into E. coli.TG1 electro-competent cells by electroporation (2500 V, 100 µs) to express

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phage displayed-scFv which constructed the Cry1F-immunized rabbit phage display library. The library

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size was evaluated by plating pooled transformants on 2×TY solid medium (contains 100 µg/mL

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ampicillin, and 1 % glucose), and its diversity evaluated by randomly collected colonies of sequence32.

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Bio-panning and isolation of anti-Cry1F toxin phage scFv colonies. The Cry1F-specific phage

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scFvs for bio-panning and isolation were performed as described in the Human Single Fold scFv

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Library (I+J) handbook (http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf)

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with some modification. In briefly, 1 mL phage particles (108 cfu/mL) of the Cry1F-immunized rabbit

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scFv library mixed with 4 mL MPBS [contains 3 % Difco™ Skim Milk in PBS buffer], then added into

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cell culture flask (25 cm²) which coated with Cry1F toxin (the first round was 80 µg/mL, the remaining

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three rounds were 60,40 and 20 µg/mL, respectively), shaking 1 h with 120 rpm after standing 1 h at

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room temperature (RT). The binding phage scFv particles were eluted by Trypsin [dissolved in PBS

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buffer (1 mg/mL)] which was used to infect fresh E. coli.TG1 that grown at 37 ℃ and be in Log phase

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(OD600=0.4), incubated at 37 ℃ for 30 min and then add KM13 helper phage (1010 cfu/mL) rescue.

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The Cry1F-specific phage scFv particles were proliferated for the next round of panning. Each round of

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enriched Cry1F-specific phage scFv particles by bio-panning were determined by polyclonal

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phage-ELISA as described by Xu et al.26. The fourth round of panned library was employed for picking 7 / 25



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randomly individual colonies and inoculated into 100 µL 2×TY liquid medium (100 µg/mL ampicillin,

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and 1 % glucose) per well of 96-well plates, then cultured with 250 rpm at 37 ℃ for overnight. Taken

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2 µL culture transfer to a new plate with 200 µL 2×TY liquid medium (100 µg/mL ampicillin, and 1 %

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glucose) from the cultured plate by hole to hole, shaking until OD600 reached to 0.4, then added helper

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phage rescue and cultured with 250 rpm at 30 ℃ for overnight. The next day, the plate was

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centrifuged for 10 min at 3 300 g at 4℃，the individual phage supernatants (100 µL) were used for

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binding Cry1F toxin and MPBS alone, then determined by monoclonal phage-ELISA as described by

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Wen et al.33.

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Colony PCR and DNA sequencing. The selected positive monoclonal phages were infected into

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E. coli.HB2151 and cultured in 2×TY liquid medium (100 µg/mL ampicillin, and 1 % glucose). Colony

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PCR reaction system was: 20 µL system contains 1 µL bacteria liquid culture, 1 µL upstream primer

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(LMB3), 1 µL downstream primer (PHEN) and its reaction condition was: 94 ℃ 10 min, then 94 ℃

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1 min, 56 ℃ 1 min, 72 ℃ 1 min for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR

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products of positive monoclonal phages scFvs were sequenced by Sangon Bio.Co.Ltd. (Shanghai,

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China) and blasted on NCBI Website.

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Soluble expression of scFv. The full length and well sequenced of positive phage clone with the

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highest activity to Cry1F toxin by monoclonal phage-ELISA was picked into 2×TY liquid medium

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(100 µg/mL ampicillin, and 1 % glucose). Shaken with 250 rpm at 37 ℃ until OD600 reached 0.8, then

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added IPTG (isopropyl-β-D-thiogalactoside, final concentration 0.8 mM) to induce the scFv soluble

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expression at 28 ℃ with 250 rpm for overnight. The next day, the culture was centrifuged for 25 min

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at 3 300 g at 4 ℃, the precipitation of cells were lysed by sonication (whole cell lysate) and TES

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buffer (periplasmic lysate) respectively. The purified soluble scFv proteins were obtained by using 8 / 25


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His-Trap HP affinity chromatography from lysates and analyzed by 12 % SDS-PAGE with silver

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staining as described by Zhang et al.28.

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IC-ELISA for Cry1F toxin based scFv. The wells of 96-well plate were coated with 100 µL 2.0

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µg/mL Cry1F toxin at 4 ℃ for standing overnight, the next day blocked by 250 µL BSA-PBS

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(contains 3 % BSA in PBS buffer) for 1.5 h at

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(contains 0.1 % Tween 20 in PBS buffer), wells were incubated with a 100 µL mixture of different

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Cry1F toxin and purified scFvs for 2 h at 28 ℃,and the concentration of Cry1F toxin contains 0.1, 0.2,

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0.5, 1, 5, 10, 50, 100, 1 000 ng/mL respectively. After washed, the bound scFvs were probed by 100 µL

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1:5 000 dilution of Anti-HIS tag [HRP] monoclonal antibodies in MPBS for 2 h at 28 ℃,then revealed

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by adding TMB solution. Waiting for color development which was performed for 15 min at RT and

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stopped with 50 µL sulphuric acid (2 M) per well. OD450 was measured by an automatic microplate

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reader. The inhibition (contains IC10, IC20, IC50 and IC80) of purified anti-Cry1F toxin scFv was

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calculated by the formula of [(P-S-N)] / (P-N)] × 100 %27, 34. P is the OD450 value of the positive

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control (50 µL purified scFv and 50 µL CBS buffer), S is the OD450 value of the standard (50 µL

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purified scFv and 50 µL serial concentration of Cry1F toxin), N is the OD450 value of the negative

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control (100 µL CBS buffer). The cross-reactivity (CR) of Cry1F toxin analogues were determined by

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the formula of [CR (%) = Cry1F (IC50) / analogues (IC50)] ×100 % according to the IC-ELISA as

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described above.

37 ℃. After washing three times with 250 µL PBST

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Assessment of the IC-ELISA for Cry1F toxin by spiked samples. The IC-ELISA was used for

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detecting Cry1F toxin standards which spiked at three different concentrations within the working

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range of detection in rice, wheat, corn and soil samples, respectively. Take rice sample as an example,

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according to the ratio of 1 g rice samples were spiked in Cry1F toxin at 10, 50, 100 ng/g concentration 9 / 25



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levels and added 1 mL 0.1 M PBS buffer (contains 0.1 % Tween 20 and 0.1 % BSA) for extraction of

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proteins, shaken with 120 rpm at RT for 2 h. The mixture of suspension were centrifuged at 10 000 g

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for 10 min and the extracts were analyzed by IC-ELISA after diluted for 10-fold with PBS buffer. The

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Cry1F toxin that spiked in wheat, corn and soil samples were operated as described above.

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Results and discussion

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Immunization of rabbit. B-cells of spleen is the animal immune system for generating antibodies,

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and their genes express antigen-specific antibodies when they are stimulated by antigen35. In order to

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obtain the Cry1F toxin specific antibodies genes from the rabbit spleen B-cells for construction of

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phage scFv library, we immunized rabbit by stepwise gradient immune method with Cry1F toxin. The

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results showed as Figure 1. The rabbit serum titer has been significantly improved after each times

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immunized by Cry1F toxin, the final titer reached 1: 5 000 000 from the strengthened immunization of

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serum, which the evaluation standard of OD450 value of Positive/Negative was over 3.0. It indicated

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that the Cry1F toxin specific antibody gene was stimulated effectively and abundantly by expressing

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antibodies, the rabbit spleen can be used for extracting total RNA to the subsequent construction of

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Cry1F toxin-immunized phage scFv library.

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Construction of Cry1F-immunized rabbit phage display scFv library. A phage display scFv

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library was constructed from the Cry1F-immunized rabbit spleen B-cells. As described, VH and VL

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cDNA were amplified (Figure 2. A), and added Linker gene turning into VL-Linker and Linker-VH

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respectively (Figure 2. B). Then, the scFv cDNA fragments were generated by SOE-PCR which

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spliced the VL-Linker and Linker-VH sequences (Figure 2. C). Final, the scFv cDNA fragments were

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added NotI and NcoI restriction sites sequences (Figure 2. D), after digestion, they were used for

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constructing pIT2-scFv recombinant phagemids which constructed the Cry1F-immunized rabbit phage 10 / 25


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display scFv library. The library capacity are 6.96×108 by calculating and each of the scFv genes has a

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real size in the agarose gel-electrophoresis by random colony PCR (Figure 2. E), which also has an

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abundant of diversity at VL (CDR1~3) and VH (CDR1~3) by sequence alignment (data not shown). In

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the process of constructed phage display scFv library, an exact quantification and purification of

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templates (contains VH, VL, VL-Linker, Linker-VH, and scFv etc.) were very essential36. Even a slight

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deviations of the equimolar ratio of templates may lead to no visible PCR products either, and

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especially in spliced VL-Linker and Linker-VH sequences by SOE-PCR. At the same time, the salt ions

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of buffer solution (such as TE buffer) also affect the outcome of PCR products, in order to avoid its

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interference, all the primers and templates were dissolved and quantified by deionized water. The

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experimental results showed that, it successfully constructed a high-quality of Cry1F-immunized rabbit

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phage display scFv library by PCR conditions performed as described above, it also would be used for

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selection of Cry1F-specific phage scFvs.

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Bio-panning and isolation of anti-Cry1F toxin phage scFv colonies. Four rounds of

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bio-panning were carried out for enrichment of anti-Cry1F toxin phage scFvs from the immunized

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rabbit phage display scFv library, and achieved the desired effect which analyzed by polyclonal

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phage-ELISA (Figure 3. A). Total 16 positive clones were obtained from the fourth rounds (Figure 3.

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B), the OD450 values of P/N (Positive/Negative) were all over 3.0. Among them, the most positive

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(named RF4) of P (1.486)/N (0.208) was 7.14, its scFv gene was sequencing and the amino acid

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sequences (translated by http://web.expasy.org/translate/) as showed in Figure 4. It also had a whole

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VH and VL of rabbit which were determined by NCBI Website (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

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Compared with the native phage display antibody library, it has a higher efficiency of antigen-specific

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phage antibodies enrichment from immunized library32, 37. Effective enrichment of antigen-binding 11 / 25



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activity of phage particles was the key prerequisites for selection of high-activity scFvs. In this study,

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we designed a series of gradient decrease in the concentration of coated Cry1F toxin in plastic cell

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culture flask, the antigen-binding activity of phage particles were captured step by step and the

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uncombined or with weak activity were washed out gradually. By this way, it can improve the

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probability and efficiency of the obtained high-activity of Cry1F toxin phage scFvs.

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IC-ELISA for Cry1F toxin based on purified RF4-scFv. E. coli.HB2151 was the host bacteria

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coordinate for soluble expression of pIT2 phagemid vector displayed-scFvs specifically, which directly

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expressing the soluble scFv proteins after infected without constructed recombinant cloned vector once

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again28. The chosen positive monoclonal of anti-Cry1F toxin rabbit phage scFv (RF4) was expressed in

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E. coli.HB2151, and obtaining purified RF4-scFv proteins with the concentration of 586.63 µg/mL by

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His-Trap HP affinity chromatography with 400 mM imidazole from periplasmic lysate (Figure 5. A).

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Converted into the original culture medium, the concentration of RF4-scFv proteins were 2.93 mg/L

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expressed in E. coli.HB2151, although better than Wan et al.27 reported 0.8 mg/L but couldn't compare

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with Lobova et al.38 reported 5.0 mg/L. Therefore, it is necessary to further optimize the expression of

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conditions, such as IPTG concentration, culture temperature and time, etc.39-41. We established the

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IC-ELISA for detecting Cry1F toxin based on purified RF4-scFv (Figure 5. B). According to the

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calculation formula of standard curve [y=10.347ln(x)+24.98, R ² =0.9702], the concentration of

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RF4-scFv binding to Cry1F toxin causing 50 % inhibition (IC50) was 11.56 ng/mL, the working

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calibration range (IC20-IC80) were between 0.92 ng/mL and 107.36 ng/mL, and the detection limit (IC10)

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was 0.18 ng/mL. The cross-reactivity (CR) of RF4-scFv to Cry1F toxin analogues were determined by

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the IC-ELISA, and showed a weakly for Cry1Ab (2.8 %), Cry1Ac (1.3 %), Cry1B, Cry1C, Cry1Ie and

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Cry2A (less than 0.1 %) as shown in Table 2, thus illustration of RF4-scFv has a strong specificity for 12 / 25


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Cry1F toxin. Compared with the Bt Cry toxins antibodies from native phage display antibody library or

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traditional immune animals of polyclonal antibody and monoclonal antibody (the IC10 were 1.5~74.0

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ng/mL)27, 28, 42-44, the RF4-scFv from Cry1F-immunized rabbit phage display scFv library not only with

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higher-activity (more than 10-fold), but also with stronger-specificity.

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IC-ELISA for Cry1F toxin spiked in samples. The practicality and applicability of RF4-scFv

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based on IC-ELISA were assessed by detection of Cry1F toxin spiked low, middle, high three different

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concentrations level at the working calibration range in rice, wheat, corn and soil samples respectively.

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The results were showed in Table 3. The mean recoveries of Cry1F toxin spiked in rice were between

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81.8 % and 96.3 % with a CV at 2.5 %~7.1 %, in wheat they were between 84.6 % and 99.6 % with a

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CV at 2.5 %~7.5 %, in corn they were between 80.2 % and 89.0 % with a CV at 2.5 %~8.0 %, in soil

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they were between 81.2 % and 95.8 % with a CV at 6.8 %~10.0 % for the intra-assay and inter-assay

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respectively. In general, the recoveries were in the range of 80.2 %~99.6 % and the CVs were among

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2.5 %~10.0 % for the spiked samples, it indicated that the RF4-scFv based on IC-ELISA had a good

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accuracy, stability and repeatability, and could be used for detection of Cry1F toxin in agro-products

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and soil samples indoor successfully, which indicates a broad application prospects for further research

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and development of Cry1F toxin rapid detection kit.

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Acknowledgments

282

The study was supported by National Natural Science Foundation of China (31630061、31371778),

283

Jiangsu Academy of Agricultural Sciences Fund Project (6111676), autonomously research project of

284

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

285

(4911406/3201604、4911406/3201608).

286

Conflicts of interest 13 / 25



287

The authors declare no conflict of interest.

288 289

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412 413 414 415 416 417 418 419 420

Figure and Table captions

OD 450

2.00 1.80

The strengthen immunization

The third immnization

1.60

The second immunization

The first immunzation

1.40

negative contorl

1.20 1.00 0.80 0.60 0.40 0.20 0.00 1

2

3

4

5

6

7

8

Dilution ratio of serum (-Lg)

421 422

Figure 1. Cry 1F toxin-immunized of rabbit serum titer were analyzed by ELISA and the OD450 values

423

were the means ± standards deviations (SD) from triplicate measurements.

424

20 / 25


Page 20 of 25

Page 21 of 25


425 426

Figure 2. The PCR amplified products of constructed rabbit phage display scFv library. (A). M: DNA

427

marker; 1: Heavy chain (VH, approximates 340 bp); 2: Light chain (VL, approximates 350 bp). (B). M:

428

DNA marker; 1: Linker-VH (approximates 390 bp); 2: VL-Linker (approximates 400 bp). (C). M: DNA

429

marker; 1-4: The whole scFv (approximates 790 bp) from VL-Linker and Linker-VH by SOE-PCR. (D).

430

scFv containing NotI and NcoI restriction sites sequences (approximates 810 bp). (E): Random of

431

colony PCR from the constructed rabbit phage scFv library (approximates 990 bp).

432 1.20 Enrichment of library

Orignal library

1.00

OD450

0.80 0.60 0.40 0.20 0.00 Round 1

Round 2

433 434

Round 3

Round 4

A 1.60

Positives

Controls

1.40

OD450

1.20 1.00 0.80 0.60 0.40 0.20

RA 3 RA 6 RA 7 RB 5 RC 2 RC 9 RD 2 RE 5 RE 8 RE 10 RF 1 RF 4 RF 9 RG 3 RG 7 RH 12

0.00

435 436

Clones

B 21 / 25



Page 22 of 25

437

Figure 3. (A) Polyclonal phage-ELISA: Each round bio-panning of anti-Cry1F toxin rabbit phage scFv

438

particles (quantified to 108 cfu/mL) were analyzed by ELISA and the OD450 values were the means ±

439

SD from triplicate measurements. (B) Monoclonal phage-ELISA: The positive monoclonal of

440

anti-Cry1F toxin rabbit phage scFv culture supernatants (quantified to 108 cfu/mL) were analyzed by

441

ELISA.

442 443

R-VL-FR1

R-VL-CDR1

R-VL-FR2

R-VL-CDR2

444 445

ELVMTQTPSSTSAAVGGTVTINCQSSQAVALMAYLSWFQQKPGQPPKLLMFGASKFASGV

446 447

PSRFKGSGSGTQFTLTISDLECADAATYYCLGGRSFDYYLWTFGGGTELVIKGGGGSGGG

R-VL-FR3 Linker -----

R-VL-CDR3

R-VH-FR1

R-VH-CDR1

R-VL-FR4

--------Linker---

R-VH-FR2

448 449

GSGGGGQEQLVESGGDLVTPGTPLTLTCTVSGLSLSHANISWVRQAPGKGLEWIGYIYPR

450 451

YSQQYARFATNGRFTISKTSTTVDLKMTSPTTEDTAYFCGRDIGYQGGSKVTRASLWGQG

452

TLVTSSGQPKAPSVTVAA

453

Figure 4. The amino acid sequences of selected positive rabbit scFv（RF4）for Cry1F toxin

R-VH-CDR2

R-VH-FR3

R-VH-CDR3

R-VH-FR4

454 455

456 457 458 459 460 461 462

A

22 / 25


Page 23 of 25


463 464 465 466 467 468

B

469

Figure 5. (A).The proteins of anti-Cry1F toxin scFv expressed in E. coli.HB2151 were analyzed by

470

SDS-PAGE. M: Protein marker; 1: Culture supernatants; 2: Whole cell lysate; 3: Periplasmic lysate;

471

4-5: Purified scFv proteins by His-Trap HP affinity chromatography with 400 mM imidazole from

472

periplasmic lysate. (B) The standard curve of purified RF4-scFv based on IC-ELISA for Cry1F toxin.

473

The valves of inhibition ratio were the means ± SD from triplicate measurements.

474 475 476 477 478 479 480 481

Table 1. The primers of used for construction of immunized rabbit phage display library（M=A or C,S=G or C, W=A or T,R=G or A）. Primer names

Nucleotide sequences (5’→3’)

RLF 1

GGGCCCAGCCGGCCGAGCTCGTGMTGACCCAGACTCCA

RLF 2

GGGCCCAGCCGGCCGAGCTCGATMTGACCCAGACTCCA

RLF 3

GGGCCCAGCCGGCCGAGCTCGTGATGACCCAGACTGAA

RLF 4

GGGCCCAGCCGGCCGAGCTCGTGCTGACTCAGTCGCCCTC

RLB 1

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATAGGATCTCCAGCTCGGTCCC

RLB 2

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATTTGACSACCACCTCGGTCCC

RLB 3

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGATTTGATTTCCAGATTGGTGCC

RLB 4

GGAAGATCTAGAGGAACCACCCCCACCACCGCCCGAGCCACCGCCACCAGAGGAGCCTGTGACGGTCAGGGTCCC

RHF 1

GGTGGTTCCTCTAGATCTTCCCAGTCGGTGGAGGAGTCCRGG

RHF 2

GGTGGTTCCTCTAGATCTTCCCAGTCGGTGAAGGAGTCCGAG

RHF 3

GGTGGTTCCTCTAGATCTTCCCAGTCGYTGGAGGAGTCCGGG

RHF 4

GGTGGTTCCTCTAGATCTTCCCAGSAGCAGCTGGWGGAGTCC

RHB

CCTGGCCCCCGAGGCCACAGTGACTGAYGGAGCCTTAGGTTGCCC

VL

VH

VL-Linker

GGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGGGAAGATCTAGAGGAACCACCCCCACC

Linker-VH

CCCGCCACCGCCGCTGCCACCTCCGCCTGAACCGCCTCCACCGGTGGTTCCTCTAGATCTTCCCAG

RF-NcoI

CATGCCATGGGGCCCAGCCGGCCGAGCTCG

Linker

scFv

23 / 25



RB-NotI

Page 24 of 25

ATAAGAATGCGGCCGCCCTGGCCCCCGAGGCCACAGTGACTGA

LMB3

CAGGAAACAGCTATGAC

PHEN

CTATGCGGCCCCATTCA

pIT2

482 483 484 485

Table 2. The cross-reactivity of RF4 purified RF4-scFv in IC-ELISA with Cry1F toxin analogues Bt Cry toxins

IC50 (ng/mL)

CR (%)

Cry1F

11.56

100

Cry1Ab

412.86

2.8

Cry1Ac

889.23

1.3

Cry1B

>8 0000

5 0000

Construction of an Immunized Rabbit Phage Display Library for

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