生物技术通报 ›› 2021, Vol. 37 ›› Issue (10): 186-195.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0197

• 研究报告 • 上一篇    下一篇

Cry1B抗独特型单链抗体的定点突变及生物活性分析

仲建锋1(), 李兴奎2, 徐重新1, 张霄1, 刘贤金1()   

  1. 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;
    2.郑州职业技术学院,郑州 450121
  • 收稿日期:2021-02-20 出版日期:2021-10-26 发布日期:2021-11-12
  • 作者简介:仲建锋,男,博士,副研究员,研究方向:农产品质量安全与生物控制技术;E-mail: jianfengzhong@jaas.ac.cn
  • 基金资助:
    国家自然科学基金项目(31630061);国家自然科学基金项目(31401784);江苏省自然科学基金项目(BK20191234)

Biological Activity of Anti-idiotypic Single Chain Fragment Variable Antibody Against Cry1B by Site-directed Mutagenesis

ZHONG Jian-feng1(), LI Xing-kui2, XU Chong-xin1, ZHANG Xiao1, LIU Xian-jin1()   

  1. 1. 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
    2. Zhengzhou Technaical College,Zhengzhou 450121
  • Received:2021-02-20 Published:2021-10-26 Online:2021-11-12

摘要:

Bt毒素的长期应用使其在害虫抗药性等方面存在生态风险,这一现状促使高特异活性和新功能Bt抗虫资源的积极开发。前期研究发现抗独特型单链抗体制备技术是开发新型杀虫蛋白的一个新途径。然而获得的Bt Cry1B抗独特型单链抗体C7与昆虫BBMV结合能力不高,需进一步改造提高。通过分子模拟技术对C7与稻纵卷叶螟BBMV上的受体氨肽酶N进行同源建模、分子对接,并预测C7与氨肽酶N结合区域的热点残基。利用C7的热点残基构建饱和突变抗体库,并采用固相筛选方法,得到突变体Y124G。BIAcore分析表明Y124G与BBMV的结合能力增加,生物测定发现对稻纵卷叶螟的杀虫活性提高。这为抗体分子改造技术的应用奠定了基础,也为新型生物农药创制提供了新的思路。

关键词: Cry1B, 分子改造, 定点突变, 杀虫活性, 表面等离子共振

Abstract:

The long-term application of Bacillus thuringiensis(Bt)toxins leads to ecological risk in pest resistance and so on,which promots the development of Bt resources with high specific activity and new functional genes. Our previous studies revealed that the preparation technique of anti-idiotypic(anti-Id)single chain fragment variable(scFv)was a new way to develop novel insecticidal protein. However,the binding ability of the obtained Bt anti-Id scFv C7 and brush-border membrane vesicles(BBMV)of Cnaphalocrocis medinalis was not high,and needed further modification and improvement. The molecular mimicking technology including homology model and molecular docking was used for C7 and the receptor aminopeptidase N in the BBMV of C. medinalis,and hot spot residues on the binding region of the C7 and the aminopeptidase N was predicted. The hot spot residues of the C7 were used to construct a saturation mutant antibody library,and solid-phase screening was to produce mutant Y124G. BIAcore analysis indicated that the binding ability of Y124G and BBMV increased. Bioassay indicated that the insecticidal activity to C. medinalis increased. This study lays the foundation for molecular modification of antibody,and provids new idea for the creation of new biological pesticides.

Key words: Cry1B, molecular modification, site-directed mutagenesis, insecticidal activity, surface plasmon resonance