密码子优化型鸭甲肝病毒VP1基因在昆虫细胞中的表达.pdfVIP

中国动物传染病学报 2014,22(1): 1-8 Chinese Journal of Animal Infectious Diseases ·研究论文· 密码子优化型鸭甲肝病毒 VP1 基因在昆虫 细胞中的表达 李传峰，陈宗艳，孟春春，梁瑞英，胡 文，刘光清 （中国农业科学院上海兽医研究所，上海200241 ） 摘 要：为了提高基因A 型鸭甲肝病毒（Duck hepatitis A virus ，DHAV ）VP1 基因在昆虫细胞中的表达水平, 本研究根据昆虫细 胞密码子偏爱性对野生型DHAV VP1 （wtVP1 ）基因进行改造, 合成了optiVP1 基因，并利用Bac-to-Bac 表达系统构建了重组杆 状病毒rBacmid-wtVP1 和rBacmid-optiVP1, 分别转染对数生长期的sf9 昆虫细胞表达VP1 蛋白。转染72 h 后，sf9 细胞出现典 型的细胞病变（cytopathic effect ，CPE ）, Western-blot 和间接免疫荧光法（indirect immunofl uorescence assay ，IFA ）检测结果表 明VP1 蛋白在重组杆状病毒感染的sf9 昆虫细胞中获得了良好表达。用Image J 软件对 Western-blot 扫描的图片进行灰度分析发现， optiVP1 基因在昆虫细胞中的表达水平明显高于wtVP1 。本研究为进一步研制诊断抗原和新型基因工程疫苗的开发奠定了基础。 关键词：鸭甲肝病毒； VP1 ；重组杆状病毒；密码子优化 中图分类号：S852.659.6 文献标志码：A 文章编号：1674-6422 （2014 ）01-0001-08 ENHANCED EXPRESSION OF CODON-OPTIMIZED VP1 GENE OF DUCK HEPATITIS A VIRUS IN INSECT CELLS LI Chuan-feng, CHEN Zong-yan, MENG Chun-chun, LIANG Rui-ying, HU Wen, LIU Guang-qing (Shanghai Veterinary Research Institute, CAAS, Shanghai 200241, China) Abstract: The objective of the present study was to enhance expression level of VP1 gene of Duck hepatitis A virus (DHAV) genotype A in insect cells by manipulating the codon usage bias. The codon usage of wild-type DHAV VP1 (wtVP1) gene was optimized and designated as optiVP1. The recombinant rBacmid-optiVP1 and rBacmid-wtVP1 plasmids were then constructed using the Bac-to-Bac baculovirus expression system (BEVS) and then transfected to sf9 insect cells at logarithmic phase for expression of VP1 protein. The typical cytopathic effect was observed in sf9 cells at 72 h post transfection. The expression of VP1 protein in sf9 cells was confi rmed in Western blotting and indirect immunofl uorescence assay (IFA). The VP1 amounts on Western blotting were measured