口蹄疫病毒非结构蛋白3ABC基因的克隆及3AB基因的原核表达  

Cloning of the Nonstructural Protein 3ABC Gene of Foot-and-Mouth Disease Virus and Expression of the 3AB Gene in E.coli

作　　者：曹轶梅[1] 刘在新[1] 卢曾军[1] 胡永浩[2] 常惠芸[1] 谢庆阁[1] 

机构地区：[1]中国农业科学院兰州兽医研究所农业部病毒学重点实验室,甘肃兰州730046 [2]甘肃农业大学,甘肃兰州730070

出　　处：《中国兽医学报》2004年第1期31-34,共4页Chinese Journal of Veterinary Science

基　　金：国家"973"计划资助项目 ( G19990 1190 1)

摘　　要：从感染口蹄疫病毒 (foot- and- m outh disease virus,FMDV)的细胞中提取总 RNA,采用反转录聚合酶链式反应(RT- PCR)获得了长度约为 14 0 0 bp的核酸片段 ,与预期长度相符。扩增产物经克隆后 ,测序证明其含有完整的 3ABC基因。该序列与国外参考序列 0 1K/ 6 6相比 ,同源性在 99%以上。亚克隆 3ABC基因至表达载体 p Tri Ex- 4 Neo中 ,通过序列分析发现 ,克隆到 p Tri Ex- 4 Neo载体上的片段于 3ABC基因 70 9～ 72 5 bp之间有 17bp的缺失 ,碰巧在 3AB基因后形成一终止密码子 ,但 3AB基因的阅读框架完整。选出含有 3AB基因完整阅读框架的阳性克隆 ,用 IPTG诱导表达 ,收集菌液进行 SDS- PAGE电泳、Western blotting分析 ,结果表明 ,3AB基因在大肠杆菌中成功表达 ,其表达产物为分子质量 33.5 ku的融合蛋白 ,并能被口蹄疫病毒阳性血清识别。经薄层扫描分析 ,表达的目的蛋白占总蛋白量的 2 6 %以上。该项研究为应用重组In this paper,first strand cDNA of 3ABC gene was synthesized using template RNA extracted from cells infected with FMDV.The complete 3ABC gene about (1 400) bp length was amplified by PCR and ligated into pGEM-T Easy vector.After transforming E.coli DH5α,ampicillin resistant colonies were isolated and plasmid DNA was prepared and analyzed by restriction analysis and PCR.Presence of the full length 3ABC gene was verified by nucleotide sequencing and the plasmid containing the expected sequence was named as pGEM-3ABC.Comparing the aquired sequence of 3ABC with that of reference strains 01K/66,the homology is more than 99%.The pGEM-3ABC was digested with SalⅠ and BglⅡ and ligated into XhoⅠ and BglⅡ-digested expression vector pTriEx-4 Neo.It was identified by sequencing that this interest fragment had a 17 bp deletion at the location of 709-725 bp of 3ABC gene,which happened to form a terminator codon behind 3AB gene,but it contained the complete open reading frame(ORF) of 3AB gene.Positive clones were selected and induced with 1 mmol/L Isopropyl-D-galactoside(IPTG),expressive host bacteria were detected by SDS-PAGE and Western blotting after properly treated.The results showed that the 3AB gene expressed successfully in E.coli and the expressed 33.5 ku fusion protein could be recognized by the bovine anti-FMDV serum.The amount of the target protein is over 26% of the total bacteria protein by gel thin layer scanning analysis.

关 键 词：口蹄疫病毒 3ABC基因 3AB基因 基因克隆 基因表达 

分 类 号：S852.65[农业科学—基础兽医学] Q78[农业科学—兽医学]