马铃薯Y病毒CI蛋白密码子优化、真核表达体系建立及单克隆抗体制备  

Codon optimization of CI protein coding sequence of potato virus Y and establishment of eukaryotic expression system and preparation of monoclonal antibody

作　　者：尹国英 贾蒙骜 常永春 张盼 李震 蔡长春 YIN Guoying;JIA Mengao;CHANG Yongchun;ZHANG Pan;LI Zhen;CAI Changchun;无(Guizhou Academ of Tobacco Science,Guiyang 550083,China;College of Agriculture,Shanxi Agricultural University,Jinzhong 030800,China;Guizhou Tobacco Company Qianxinan Prefecture Company,Xingyi 562400,China;Hubei Academy of Tobacco Science,Wuhan 430030,China)

机构地区：[1]贵州省烟草科学研究院,贵阳550081 [2]山西农业大学农学院,晋中030800 [3]贵州省烟草公司黔西南州公司,兴义562400 [4]湖北省烟草科学研究院,武汉430030

出　　处：《植物病理学报》2024年第3期544-554,共11页Acta Phytopathologica Sinica

基　　金：国家自然科学基金项目(32160631);中国烟草总公司贵州省公司科技项目(2022XM08);中国烟草总公司贵州省公司科技项目(2021XM08);贵州省烟草科学研究院科技项目(GZYKY2021-02);贵州省科技计划项目([2020]4102)。

摘　　要：编码马铃薯Y病毒(potato virus Y,PVY)CI蛋白的核酸序列具有多个类似原核生物启动子元件的区域,有可能在原核细胞中翻译出对原核细胞具有毒性的蛋白,因此该序列难以按照常规的试验方法进行载体构建。在不改变CI氨基酸序列的基础上,对编码CI的核酸序列进行改造,成功构建了含有CI开放阅读框(ORF)的表达载体,利用真核无细胞蛋白表达系统成功实现了CI蛋白的表达。通过亲和层析纯化,将CI蛋白免疫Balb/c小鼠,共制备了6株单克隆抗体。间接ELISA和Western blot免疫印迹证明,其中4B7_2D6(IgG1)单克隆抗体,灵敏度最高,特异性最强。CI蛋白的成功表达及单克隆抗体的获得,为后续研究CI蛋白的结构与功能提供了前提条件,对进一步探索CI与植物蛋白互作机理以及PVY的检测技术具有重要意义。The nucleic acid sequence encoding the CI protein of potato virus Y(PVY)had multiple regions similar to prokaryotic promoter elements.It was possible to translate toxic proteins in prokaryotic cells,so it was difficult to construct vectors.According to the codon bias of prokaryotes,the CI sequence was modified without changing the amino acid sequence.The expression vector containing CI open reading frame(ORF)was successfully constructed,and CI protein was successfully expressed through the eukaryotic cell-free protein expression system.Six cell lines were prepared by immunizing Balb/c mice with purified protein using hybridoma technology.Indirect ELISA and western blot showed that the prepared CI monoclonal antibody 4B7_2D6(IgG1)had high sensitivity and specificity.The successful expression of CI protein provides a prerequisite for the purification of CI protein and the subsequent study of the structure and function of CI protein,which is of great significance for further exploring the interaction mechanism between CI and plant proteins.

关 键 词：CI蛋白 密码子偏好性 真核表达 单克隆抗体 

分 类 号：S432.41[农业科学—植物病理学]