靶向模拟Bt Cry1C蛋白抗虫功能的人源化基因工程抗体筛选及鉴定

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

摘要/Abstract

摘要：

Bt蛋白制剂及其转基因作物长时间推广应用,害虫抗药性等问题日益凸显,探索新型安全抗虫材料已成为竞先研究的热点。基于抗体免疫网络学说的抗独特型抗体技术被证实可用于研发抗原生物活性类似物,有望成为靶向创制模拟Bt蛋白抗虫功能材料的新途径。以Bt Cry1C蛋白多克隆抗体为包被抗原,从人源化噬菌体单链抗体库中获得9个阳性单克隆,其中E8-Anti-Id scFv经鉴定为β型抗独特型基因工程单链抗体。Bt Cry1C蛋白多克隆抗体、小菜蛾（Plutella xylostella）刷状缘膜囊泡（brush border membrane vesicles,BBMV）蛋白和稻纵卷叶螟（Cnaphalocrocis medinalis）BBMV蛋白对E8-Anti-Id scFv的竞争抑制中浓度（IC₅₀）分别为2.625、3.638和4.605 μg/mL。室内生物测定显示,噬菌体展示形式的E8-Anti-Id scFv在滴度为1.2×10⁸ CFU/mL浓度条件下,对供试的小菜蛾和稻纵卷叶螟在72 h内的校正死亡率为58.69%和52.82%,其抗虫活性分别达到浓度为20 μg/mL的原Bt Cry1C蛋白的77.87%和73.21%。由此表明,获得的E8抗独特型基因工程单链抗体材料初步具备模拟Bt Cry1C蛋白抗虫功能的特性,为后期深入研究和推进应用奠定了技术和材料基础。

关键词: Bt Cry1C蛋白, 抗独特型抗体, 噬菌体抗体库, 基因工程抗体

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

徐重新, 张霄, 刘媛, 仲建锋, 谢雅晶, 卢莉娜, 高美静, 刘贤金. 靶向模拟Bt Cry1C蛋白抗虫功能的人源化基因工程抗体筛选及鉴定[J]. 生物技术通报, 2022, 38(5): 191-200.

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.

图/表 9

表1 各轮次生物淘筛过程中噬菌体颗粒的投入和产出

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

图1 各轮次富集噬菌体颗粒对Bt Cry1C蛋白多克隆抗体的多克隆噬菌体ELISA

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

图2 阳性克隆对Bt Cry1C蛋白多克隆抗体的单克隆噬菌体ELISA

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

图3 阳性单克隆人源单链抗体基因PCR扩增产物

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

图4 阳性单克隆人源单链抗体基因对应氨基酸序列比对

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

图5 基于间接竞争ELISA法的E8-Anti-Id scFv分型测定

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

图6 基于间接竞争ELISA法的E8-Anti-Id scFv竞争抑制率测定

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

图7 E8-Anti-Id scFv在48 h内对稻纵卷叶螟和小菜蛾室内杀虫效果 A：供试虫子为稻纵卷叶螟（CK-为PBS缓冲液;CK+为溶于PBS缓冲液中的Bt Cry1C蛋白溶液;E8为溶于PBS溶液缓冲液中的噬菌体展示形式的E8-Anti-Id scFv溶液）;B：供试虫子为小菜蛾（CK-为PBS缓冲液;CK+为溶于PBS缓冲液中的Bt Cry1C蛋白溶液;E8为溶于PBS溶液中的噬菌体展示形式的E8-Anti-Id scFv溶液）

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）

表2 E8-Anti-Id scFv对小菜蛾和稻纵卷叶螟室内杀虫活性

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

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