CryNGc蛋白在转基因玉米和大肠杆菌中表达的等同性研究  

Equivalence Study of CryNGc Protein Expressed in Transgenic Maize and Escherichia coli

作　　者：赵方方[1] 刘洋[2] 井乐刚[1] 尹悦佳[2] 冯树丹[1] 刘相国[2] ZHAO Fangfang;LIU Yang;JING Legang;YIN Yuejia;FENG Shudan;LIU Xiangguo(College of Life Science and Technology, Harbin Normal University, Harbin 150025, China;Jilin Provincial Key Laboratory of Agricultural Biotechnology, Institute of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences Changehun 130033, China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院,哈尔滨150025 [2]吉林省农业科学院农业生物技术研究所吉林省农业生物技术重点实验室,长春130033

出　　处：《生物技术进展》2018年第1期41-45,共5页Current Biotechnology

基　　金：国家转基因生物新品种培育重大专项(2015ZX8013002-007);吉林省农业创新工程(CXGC2017ZY026)资助

摘　　要：转基因玉米表达蛋白的身份鉴定和对玉米表达蛋白与大肠杆菌表达蛋白的等同性研究是进行蛋白安全性评价的重要前提。以人工合成的新型重组抗虫蛋白CryNGc为研究材料,通过密码子优化、cryNGc杀虫蛋白编码基因化学合成、原核表达载体构建、大肠杆菌表达条件优化等方法原核表达CryNGc蛋白。在此基础上,利用SDS-PAGE电泳、免疫杂交分析和液相色谱-串联质谱鉴定技术对玉米转化体HiII-NGc-1和大肠杆菌表达的CryNGc重组蛋白进行了特性分析和一致性评价。研究结果表明,抗虫基因cryNGc在转基因玉米和大肠杆菌中表达的蛋白电泳迁移率、免疫活性和氨基酸组成具有一致性,证实玉米转化体HiII-NGc-1中产生的CryNGc蛋白的特性与理论预测相符,且与大肠杆菌表达的CryNGc蛋白实质等同。研究结果为进一步开展新型抗虫重组蛋白CryNGc玉米转化体的生物安全性评价提供了理论基础和数据支持。The important precondition of safety evaluation of protein in genetically engineered maize is protein identification and equivalence study of proteins expressed in genetically engineered maize and Escherichia coli. CryNGc protein was expressed in E. coli through optimized methods such as codon optimization,cryNGc insecticidal protein gene synthesis,prokaryotic expression vector construction and E. coli expression conditions optimizition. On this basis,the characteristics and consistency of the recombinant protein Hi II-NGc-1 in maize and CryNGc expressed in E. coli were analyzed by SDS-PAGE,immunoblot analysis and liquid chromatography-tandem mass spectrometry. The results showed that there was consistent in the expression of cryNGc gene in transgenic maize and E. coli by electrophoresis mobility,immune competence and amino acid composition consistent. The results confirmed that the CryNGc protein characteristics of maize transformants HiII-NGc-1 was consistent with the theoretical prediction,and was substantially equivalent to the CryNGc protein expressed in E. coli. The results provided the theoretical basis and data support for further biosafety evaluation of the new insect-resistant recombinant protein CryNGc maize transformants.

关 键 词：抗虫蛋白 cryNGc基因 原核表达 等同性 转基因 

分 类 号：Q943.2[生物学—植物学] S513[农业科学—作物学]