Screening and Identification of Humanized Genetically Engineered Antibody Targeting to Simulate the Anti-insect Function of Bt Cry1C Protein

doi:10.13560/j.cnki.biotech.bull.1985.2021-0991

Abstract

Abstract:

It has become a hot spot to explore new safe insect-resistant materials,as the pest resistance problem being increasingly prominent alongside the promotion and application of Bt protein and its transgenic crops. The anti-idiotype antibody technology based on antibody immune network theory has been proved to be useful for antigenic bioactive analogue research,and is expected to be a new approach for the targeted creation of the materials simulating the anti-insect function of Bt protein. Using Bt Cry1C protien polyclonal antibody as coated antigen,there were nine positive monoclone obtained from a humanized phage single-chain antibody library. Among them,the E8-Anti-Id scFv was identified as a β type anti-idiotypic genetically engineered single-chain antibody. The half-maximum inhibition concentrations（IC₅₀）of Bt Cry1C polyclonal antibodies（pAbs）,Plutella xylostella Brush border membrane vesicles（BBMV）and Cnaphalocrocis medinalis BBMV to E8-Anti-Id scFv were 2.625,3.638 and 4.605 μg/mL,respectively. Laboratory bioassay showed that the corrected mortality of C. medinalis BBMV and P. xylostella BBMV at 72 h were 58.69% and 52.82% at the phage-displayed E8-Anti-Id scFv of titer 1.2×10⁸CFU/mL,respectively. Its anti-insect activity reached 77.87% and 73.21% of original Bt Cry1C protein（20 μg/mL）,respectively. These results indicate that the E8-Anti-Id scFv has preliminary characteristics of mimics the insect-resistant function of Bt Cry1C protein,which lays a technical and material foundation for further research and application.

Key words: Bt Cry1C protein, anti-idiotype antibody, phage antibody library, genetically engineered antibody

XU Chong-xin, ZHANG Xiao, LIU Yuan, ZHONG Jian-feng, XIE Ya-jing, LU Li-na, GAO Mei-jing, LIU Xian-jin. Screening and Identification of Humanized Genetically Engineered Antibody Targeting to Simulate the Anti-insect Function of Bt Cry1C Protein[J]. Biotechnology Bulletin, 2022, 38(5): 191-200.

Figures/Tables 9

Table 1 Input and output of phage particles in each round of biopanning

筛选轮数 Biopanning round	抗原包被浓度 Concentration of coated antigen/（μg·mL^-1）	投入噬菌体 Input of phage particles/（μg·mL^-1）	产出噬菌体 Output of phage particles/（μg·mL^-1）	投入产出比 Bound of phage particles/%
1	100	5.65×10¹⁰	3.32×10⁶	5.88×10^-3
2	50	4.91×10¹⁰	2.09×10⁷	4.26×10^-2
3	25	6.62×10¹⁰	1.96×10⁹	2.96
4	10	7.06×10¹⁰	3.12×10⁹	4.42

Fig. 1 Polyclonal phage ELISA of each round of enriched library phage particles for Bt Cry1C pAbs（Poly-clonal antibodies）

Fig. 2 Monoclonal phage ELISA of positive clones for Bt Cry1C pAbs

Fig. 3 PCR amplified products of positive monoclonal human scFvs genes

Fig. 4 Amino acid sequences aligment of positive monoclonal human scFvs genes

Fig. 5 Determination of the type of E8 -Anti-Id scFv based on IC-ELISA

Fig. 6 Determination of the competitive inhibition of E8 -Anti-Id scFv based on IC-ELISA

Fig. 7 Insecticidal effects of E8-Anti-Id scFv to C. medinalis and P. xylostella at 48 h indoor A：Insect used in the experiment is C. medinalis（CK- is PBS buffer. CK+ is Bt Cry1C protein solution dissolved in PBS buffer. E8 is phage-displayed E8-Anti-Id scFv solution dissolved in PBS buffer）. B：Insect used in the experiment is P.xylostella（CK- is PBS buffer. CK+ is Bt Cry1C protein solution dissolved in PBS buffer. E8 is phage-displayed E8-Anti-Id scFv solution dissolved in PBS buffer）

Table 2 Insecticidal activities of E8-Anti-Id scFv to C. medinalis and P. xylostella indoor

供试样品 Samples for bioassay	稻纵卷叶螟校正死亡率Corrected mortality for C. medinalis/%			小菜蛾校正死亡率 Corrected mortality for P. xylostella/%
供试样品 Samples for bioassay	24 h	48 h	72 h	24 h	48 h	72 h
CK-（PBS solution）	0.00±0.00a	0.00±0.00a	3.52±1.26a	0.00±0.00a	0.00±0.00a	1.36±2.01a
CK+（Bt Cry1C protein）	37.21±4.26a	65.56±3.77a	72.15±1.36a	35.16±1.12a	69.25±4.13a	75.36±1.29a
E8（E8-Anti-Id scFv）	29.83±3.13a	44.32±2.09a	52.82±4.79a	26.38±1.01a	48.99±1.23a	58.69±2.32a

References 24

[1]	Chandler D, Bailey AS, Tatchell GM, et al. The development, regulation and use of biopesticides for integrated pest management[J]. Philos Trans R Soc Lond B Biol Sci, 2011, 366(1573):1987-1998. doi: 10.1098/rstb.2010.0390 URL
[2]	Bravo A, Likitvivatanavong S, Gill SS, et al. Bacillus thuringiensis:a story of a successful bioinsecticide[J]. Insect Biochem Mol Biol, 2011, 41(7):423-431. doi: 10.1016/j.ibmb.2011.02.006 URL
[3]	国际农业生物技术应用服务组织. 2019年全球生物技术/转基因作物商业化发展态势[J]. 中国生物工程杂志, 2021, 41(1):114-119.
	International Service for the Application of Agricultural Biotechnology. Global commercialization trend of biotechnology/GM crops in 2019[J]. China Biotechnol, 2021, 41(1):114-119.
[4]	Jurat-Fuentes JL, Heckel DG, Ferré J. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis[J]. Annu Rev Entomol, 2021, 66:121-140. doi: 10.1146/annurev-ento-052620-073348 pmid: 33417820
[5]	Abbas MST. Genetically engineered(modified)crops(Bacillus thuringiensis crops)and the world controversy on their safety[J]. Egypt J Biol Pest Control, 2018, 28(1):1-12. doi: 10.1186/s41938-017-0002-3 URL
[6]	徐重新, 张霄, 刘媛, 等. 抗独特型抗体在食用农产品危害物监控中的应用研究[J]. 食品科学技术学报, 2019, 37(4):103-110.
	Xu CX, Zhang X, Liu Y, et al. Study on anti-idiotype antibodies used for monitoring hazards in edible agricultural products[J]. J Food Sci Technol, 2019, 37(4):103-110.
[7]	Hanoux V, Wijkhuisen A, Alexandrenne C, et al. Polyclonal anti-idiotypic antibodies which mimic an epitope of the human prion protein[J]. Mol Immunol, 2009, 46(6):1076-1083. doi: 10.1016/j.molimm.2008.09.033 URL
[8]	Lan HN, Zheng X, Khan MA, et al. Anti-idiotypic antibody:a new strategy for the development of a growth hormone receptor antagonist[J]. Int J Biochem Cell Biol, 2015, 68:101-108. doi: 10.1016/j.biocel.2015.09.004 URL
[9]	Shu M, Xu Y, Liu X, et al. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins:Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal[J]. Anal Chim Acta, 2016, 924:53-59. doi: 10.1016/j.aca.2016.03.053 URL
[10]	Qiu Y, Li P, Dong S, et al. Phage-mediated competitive chemiluminescent immunoassay for detecting Cry1Ab toxin by using an anti-idiotypic camel nanobody[J]. J Agric Food Chem, 2018, 66(4):950-956. doi: 10.1021/acs.jafc.7b04923 URL
[11]	徐重新, 杨晶祎, 陆梦晓, 等. 间接竞争时间分辨荧光免疫分析法检测稻米中Cry1C毒素[J]. 南京农业大学学报, 2014, 37(6):44-48.
	Xu CX, Yang JY, Lu MX, et al. Indirect competitive TRFIA(CI-TRFIA)method development for the determination of Cry1C toxin in rice[J]. J Nanjing Agric Univ, 2014, 37(6):44-48.
[12]	Lee CM, Iorno N, Sierro F, et al. Selection of human antibody fragments by phage display[J]. Nat Protoc, 2007, 2(11):3001-3008. doi: 10.1038/nprot.2007.448 URL
[13]	Zhang X, Liu Y, Zhang CZ, et al. Rapid isolation of single-chain antibodies from a human synthetic phage display library for detection of Bacillus thuringiensis(Bt)Cry1B toxin[J]. Ecotoxicol Environ Saf, 2012, 81:84-90. doi: 10.1016/j.ecoenv.2012.04.021 URL
[14]	Xu C, Liu X, Zhang C, et al. Establishment of a sensitive time-resolved fluoroimmunoassay for detection of Bacillus thuringiensis Cry1Ie toxin based nanobody from a phage display library[J]. Anal Biochem, 2017, 518:53-59. doi: 10.1016/j.ab.2016.11.006 URL
[15]	仲建锋, 李兴奎, 徐重新, 等. Cry1B抗独特型单链抗体的定点突变及生物活性分析[J]. 生物技术通报, 2021, 1-10.
	Zhong Jianfeng, Li Xingkui, Xu Chongxin, et al. Enhanced insecticide activity of anti-idiotypic single chain Fragment variable antibody against Cry1B by site-directed mutagenesis[J]. Biotechnol Bulletin, 37(10):186-195.
[16]	Ossysek K, Uchański T, Kulesza M, et al. A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I+J phagemid libraries[J]. Immunol Lett, 2015, 167(2):95-102. doi: 10.1016/j.imlet.2015.07.005 URL
[17]	Sokolowska-Wedzina A, Chodaczek G, Chudzian J, et al. High-affinity internalizing human scFv-fc antibody for targeting FGFR1-overexpressing lung cancer[J]. Mol Cancer Res, 2017, 15(8):1040-1050. doi: 10.1158/1541-7786.MCR-16-0136 pmid: 28483948
[18]	de la Cruz S, Cubillos-Zapata C, López-Calleja IM, et al. Isolation of recombinant antibody fragments(scFv)by phage display technology for detection of almond allergens in food products[J]. Food Control, 2015, 54:322-330. doi: 10.1016/j.foodcont.2015.02.011 URL
[19]	Dong S, Bo Z, Zhang C, et al. Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library[J]. Appl Microbiol Biotechnol, 2018, 102(7):3363-3374. doi: 10.1007/s00253-018-8797-8 pmid: 29484477
[20]	Hao J, Li YH, Wang JX, et al. Screening and activity identification of an anti-idiotype nanobody for Bt Cry1F toxin from the camelid naive antibody phage display library[J]. Food Agric Immunol, 2020, 31(1):1-16. doi: 10.1080/09540105.2019.1691156 URL
[21]	Xu C, Liu X, Liu Y, et al. High sensitive single chain variable fragment screening from a microcystin-LR immunized mouse phage antibody library and its application in immunoassay[J]. Talanta, 2019, 197:397-405. doi: 10.1016/j.talanta.2019.01.064 URL
[22]	Zhao AZ, Tohidkia MR, Siegel DL, et al. Phage antibody display libraries:a powerful antibody discovery platform for immunotherapy[J]. Crit Rev Biotechnol, 2016, 36(2):276-289. doi: 10.3109/07388551.2014.958978 URL
[23]	Xie Y, Xu C, Gao M, et al. Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth(Plutella xylostella)[J]. Pest Manag Sci, 2021, 77(10):4593-4606. doi: 10.1002/ps.6499 URL
[24]	Simons JF, Lim YW, Carter KP, et al. Affinity maturation of antibodies by combinatorial Codon mutagenesis versus error-prone PCR[J]. MAbs, 2020, 12(1):1803646. doi: 10.1080/19420862.2020.1803646 URL