苏云金芽孢杆菌Cry毒素共性结构短肽串联表达及功能分析  

Tandem Expression and Functional Analysis of Common Structure Short Peptides of Bacillus thuringiensis Cry Toxins

作　　者：金嘉凤 沈成 孟萌 陈蔚[2] 徐重新 刘媛[1,2] 刘贤金 JIN Jiafeng;SHEN Cheng;MENG Meng;CHEN Wei;XU Chongxin;LIU Yuan;LIU Xianjin(School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base of Ministry of Science and Technology/Institute of Food Safety and Nutrition,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;College of Plant Protection,Nanjing Agricultural University,Nanjing 210023,China;School of Life Sciences,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学食品与生物工程学院,江苏镇江212013 [2]江苏省农业科学院农产品质量安全与营养研究所/省部共建国家重点实验室培育基地—江苏省食品质量安全重点实验室,江苏南京210014 [3]南京农业大学植物保护学院,江苏南京210023 [4]江苏大学生命科学学院,江苏镇江212013

出　　处：《河南农业科学》2024年第1期96-104,共9页Journal of Henan Agricultural Sciences

基　　金：江苏省自然科学基金面上项目(BK20231384);国家自然科学基金项目(31972292);江苏省农业科技自主创新资金项目[CX(22)1009)]。

摘　　要：苏云金芽孢杆菌(Bacillus thuringiensis,Bt)Cry毒素是一类具有抗虫功能的微生物蛋白。将前期基于Bt Cry毒素家族氨基酸序列及结构信息设计获得的呈现Cry毒素共性结构的3个短肽,拼接成为具有免疫原性的长链多肽(Bt Cry-GXJG-11),以期研发Bt Cry毒素杀虫功能的模拟物及制备Bt Cry毒素检测用广谱抗体的免疫原。分子建模预测结果显示,Bt Cry-GXJG-11与Bt Cry毒素靶标害虫棉铃虫(Helicoverpa armigera)钙黏蛋白受体(HaCad-TBR)存在互作效应,并初步明确了两者互作的关键氨基酸位点为244TYR。经原核表达,获得了质量浓度为0.5 mg/mL的Bt Cry-GXJG-11纯蛋白,测得其与HaCad-TBR的亲和力常数(KD)为2.151×10^(-7)mol/L。室内生测试验表明,Bt Cry-GXJG-11对棉铃虫具有一定杀虫活性,校正死亡率为27.5%;且以其免疫的小鼠血清能同时识别6种Bt Cry毒素(Cry1Ab、Cry1Ac、Cry1Ah、Cry1B、Cry1C、Cry1F)。Bacillus thuringiensis(Bt)Cry toxins are a class of microbial proteins with insecticidal activities.Previously,based on the amino acid sequence and structural information of the Bt Cry toxin family,three short peptides presenting the common structure of Cry toxins were designed and obtained.These peptides were spliced to be a polypeptide with immunogenicity(Bt Cry-GXJG-11),which was potentially to be used to design novel insecticidal mimics of Bt Cry toxins,and to produce broad specificity antibodies against Bt Cry toxins as the immunogen.Molecular modeling showed that Bt Cry-GXJG-11 was potentially interacting with HaCad-TBR,which was a recombinant expressed receptor of Cry toxins in Helicoverpa armigera and the contacting amino acids(244TYR)in the complex were also predicted.After prokaryotic expression,the purified Bt Cry-GXJG-11 was obtained at a concentration of 0.5 mg/mL,and its affinity constant(KD)with HaCad-TBR was measured to be 2.151×10^(-7) mol/L.The bioassay showed that Bt Cry-GXJG-11 had weak insecticidal activity against H.armigera,with the mortality of 27.5%.In addition,the antiserum of mice immunized with Bt Cry-GXJG-11 could simultaneously recognize six Cry toxins(Cry1Ab,Cry1Ac,Cry1Ah,Cry1B,Cry1C,Cry1F).

关 键 词：Bt Cry毒素 共性结构 分子互作 杀虫活性 广谱抗体 棉铃虫 

分 类 号：Q965.9[生物学—昆虫学]