伪狂犬病病毒鄂A株gC基因的克隆、序列分析及其在大肠杆菌中的表达  

Cloning ,Sequening Analysis of the gC Gene of Pseudorabies Virus Strain Ea and Its Expression in E.coli

作　　者：肖少波[1] 陈焕春[1] 方六荣[1] 洪文洲[1] 何启盖[1] 马相如[1] 

机构地区：[1]华中农业大学牧医学院动物病毒室,武汉430070

出　　处：《中国农业科学》2002年第2期202-206,共5页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目 (3 9670 5 5 53 9970 5 5 9)

摘　　要：以伪狂犬病病毒国内地方分离株 (鄂A株 )为材料 ,采用核酸杂交、DNA重组技术直接从基因组DNA中克隆了最主要的保护性抗原基因gC ,测定了全序列并对其原核表达产物作为血清学诊断抗原进行了初步探讨。结果表明 :所克隆的 gC基因全长 175 5bp(含启动子序列和 poly(A)信号序列 ) ,具有典型I型膜蛋白结构特征。同具有代表性的国外标准株NIA 3株、IndianaS株相比 ,多个酶切点不同 ,尤其是具有分型意义的BamHI位点消失 ;氨基酸水平上也存在一定程度的差异 ,鄂A株 gC可编码 4 87个氨基酸残基 ,而以往所报道的gC均只编码 4 78或 4 79个氨基酸 ,有意义的是功能区突变较多 ,并存在连续 7个氨基酸残基的插入。同 pET 2 8a的 6xHis Tag融合的gC基因完整编码区在大肠杆菌中获得高效表达 ,融合蛋白分子量为 6 2ku ,能同伪狂犬病病毒高免血清发生特异性反应。纯化的表达产物作为ELISA和乳胶凝集诊断抗原 。The glycoprotein C(gC) gene of Pseudorabies virus(PRV) strain Ea was cloned, sequenced, and the expression products in E.coli were studied to establish a safe, specific and sensitive diagnosis method.The results showed that the complete gC gene of PRV strain Ea was 1755bp(s) from promoter to ploy(A) signal sequences, existing mutation in BamHI, SacI, SalI and SmaI sites and multipile site mutations and one insert mutation, especially in the function domain when compared with the PRV strains NIA 3 and Indiana S. The expression products of the gC, which fused with 6xHis Tag of the vector pET 28a was 62ku and specific to antisera against PRV Ea strain by Western blotting. Purified fusion protein was specific and sensitive as the antigen of indirect ELISA and Latex Agglutination Test(LAT).

关 键 词：伪狂犬病病毒 鄂A株 GC基因 克隆 序列分析 基因表达 

分 类 号：S852.65[农业科学—基础兽医学]