动物源化改型单链抗体的设计、表达及杀虫活性  

Design,expression,and insecticidal activity of novel animalized single chain variable fragments by CDR grafting

作　　者：仲建锋[1] 高美静 卢莉娜 张志勇[1] ZHONG Jianfeng;GAO Meijing;LU Lina;ZHANG Zhiyong(Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,Key Laboratory for Control Technology and Standard for Agro-product Safety and Quality,Ministry of Agriculture and Rural Affairs,Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省食品质量安全重点实验室——省部共建国家重点实验室培育基地,农业农村部农产品质量安全控制技术与标准重点实验室,江苏省农业科学院农产品质量安全与营养研究所,南京210014

出　　处：《农药学学报》2024年第6期1069-1079,共11页Chinese Journal of Pesticide Science

基　　金：国家自然科学基金(32272589);科技创新2030重大项目(2022ZD0402007);江苏省自然科学基金(BK20210155)。

摘　　要：抗独特型抗体制备技术是开发新型抗虫蛋白资源的一项创新策略。为设计构造以基因工程抗体为基础的杀虫蛋白资源,制备了对小菜蛾(Plutella xylostella)具有杀虫活性的猪、牛源改型单链抗体(single chain variable fragment,sc Fv)。通过NCBI数据库搜索猪、牛源抗体重、轻链可变区的8个骨架区,在其中植入人源抗独特型sc Fv 3E1的6个互补决定区,并进行人工合成及替换成噬菌体展示表达载体p IT2;利用间接竞争ELISA对动物源化改型sc Fv进行分型鉴定;利用表面等离子共振(surface plasmon resonance,SPR)技术分析动物源化改型sc Fv与小菜蛾刷状缘膜囊泡(brush border membrane vesicles,BBMV)的结合能力;采用浸叶法测定动物源化改型sc Fv对小菜蛾的生物活性。通过替换表达载体获得了猪源swine-3E1-p IT2质粒和牛源bovine-3E1-p IT2质粒,结果显示:swine-p IT2和bovine-p IT2表达上清液对Cry1B与其多克隆抗体(p Ab)结合的抑制率分别为44.3%和43.0%,模拟抗原Cry1B的某些结构功能,发现两者均属于β型抗独特型sc Fv。SPR分析发现,swine-p IT2、bovine-p IT2与小菜蛾BBMV的结合能力分别为209.48和195.31 RU。生物活性测定结果显示,改造后的swinep IT2和bovine-p IT2对小菜蛾幼虫均具有杀虫活性,LD_(50)值分别为5.90×10^(7)和6.22×10^(7)CFU/m L,均低于亲本3E1的6.86×10^(7)CFU/m L;并且改造后2种材料的毒力回归方程的斜率分别为1.50和1.48,均高于3E1(1.23)。综上,动物源化swine-p IT2和bovine-p IT2改型sc Fv属β型抗独特型sc Fv,能够与小菜蛾的BBMV结合,且具有杀虫活性,为新型生物农药蛋白资源挖掘提供了新的思路。The preparation of anti-idiotypic antibodies is an innovative strategy for developing novel insecticidal protein resources.To design and construct insecticidal protein resources by engineering antibody technology,two animalized single-chain variable fragments(scFv)against Plutella xylostella larvae were constructed by CDR grafting.The amino acid sequences of framework regions(FRs)in the heavy chain variable(VH)and light chain variable(VL)of the swine and bovine antibodies were obtained from the NCBI database.Subsequently,the complementarity determining regions(CDRs)of the humanized anti-idiotypic scFv 3E1 were grafted onto the FRs of the swine and bovine antibodies,respectively.The reconstructed sequence was analyzed and synthesized,and the phage displaying the pIT2 plasmid was acquired by substituting the pUC57 vector.Moreover,an indirect competitive enzyme-linked immunosorbent assay(ELISA)was employed to typify and characterize the CDR grafting scFv.The binding interaction between the animalized scFv and the brush border membrane vesicles(BBMV)of P.xylostella was analyzed using surface plasmon resonance(SPR)analysis.Furthermore,the biological activity of the animalized scFv with grafted CDRs against P.xylostella was determined by the leaf dipping assay.The results showed that both the swine-3E1-pIT2 and bovine-3E1-pIT2 plasmids were obtained by replacing the original vector PUC57.Furthermore,both the swinepIT2 and bovine-pIT2 exhibited 44.3%and 43.0%inhibition of the bindings of Cry1B to its polyclonal antibody,respectively.The results indicated that both the swine-pIT2 and bovine-pIT2 could be classified asβsubtype anti-idiotypic scFvs,which could simulate the partial structural features of Cry1B.Subsequently,the binding affinities of swine-pIT2 and bovine-pIT2 with P.xylostella BBMV were determined by SPR analysis to be 209.48 and 195.31 RU,respectively.The bioassay results indicated that swine-pIT2 and bovine-pIT2 exhibited insecticidal activity against P.xylostella larvae.Both LD_(50)values of the swine-pI

关 键 词：动物源化 BT毒素 改型抗体 杀虫活性 表面等离子共振 小菜蛾 

分 类 号：S482.39[农业科学—农药学]