密码子优化型鸭甲肝病毒VP1基因在昆虫细胞中的表达  

ENHANCED EXPRESSION OF CODON-OPTIMIZED VP1 GENE OF DUCK HEPATITIS A VIRUS IN INSECT CELLS

作　　者：李传峰[1] 陈宗艳[1] 孟春春[1] 梁瑞英[1] 胡文[1] 刘光清[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2014年第1期1-8,共8页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金项目(31101848);中央级公益性科研院所基本科研业务费专项(2012JB13);公益性行业(农业)科研专项(201003012);国家高技术研究发展计划(863)项目(2011AA10A200)

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。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 confirmed in Western blotting and indirect immunofluorescence assay （IFA）. The VP1 amounts on Western blotting were measured using the software Image J. The expression level of optiVP1 gene was significantly increased as compared with wtVP1 gene. This study provided a basis for development of diagnostic reagents and genetically engineered novel vaccines for DHAV.

关 键 词：鸭甲肝病毒 VP1 重组杆状病毒 密码子优化 

分 类 号：S852.659.6[农业科学—基础兽医学]