山羊痘病毒p32基因的原核表达及生物信息分析  

Prokaryotic Expression and Bioinformatics Analysis of Goatpox Virus p32 Gene

作　　者：蒲德治 龚晨燕 刘永[2] 蒋高建 于峰[2] 

机构地区：[1]江苏大学食品科学与生物工程学院,江苏镇江212013 [2]江苏大学生命科学学院,江苏镇江212013

出　　处：《生物技术》2012年第4期32-37,共6页Biotechnology

基　　金：江苏大学高级人才基金资助项目(1281330017);江苏省自然科学基金项目(BK2010328)资助

摘　　要：目的:构建表达山羊痘(GPV)P32蛋白的原核表达载体PTYB11-gpvp32,初步分析其在大肠杆菌(E.coli)BL21中的较佳表达条件。方法:首先用序列分析软件Blastp、MEGA5.1、SOSUI等对GPVP32进行生物信息学分析;Primer Primier5.0软件设计引物,在上游引物添加限制性酶切位点SpeⅠ、下游引物添加限制性酶切位点XhoⅠ,以pSK-gpvp32为模板,PCR扩增方法获得基因片段,克隆入PMD18-Simple-T,构建PMD18SimpleT-gpvp32载体,转化大肠杆菌(E.coli)Top10感受态细胞,经酶切筛选、测序确定插入序列的正确,然后将gpvp32克隆入原核表达载体PTYB11中的SpeⅠ、XhoⅠ酶切位点,构建原核表达质粒PTYB11-gpvp32;转化大肠杆菌(E.coli)BL21后,IPTG诱导重组蛋白表达,SDS-PAGE分析和Western-blot鉴定重组蛋白在不同诱导时间下的表达情况。结果:成功构建山羊痘病毒原核表达载体PTYB11-gpvp32并在大肠杆菌(E.coli)BL21中成功表达GPVP32,经检测重组蛋白表达量约为菌体总蛋白的28%,进化树结果表明山羊痘的亲缘关系与地理位置存在差异及在不同的羊群中进化的结果,经SOSUI等软件综合分析知GPVP32是山羊痘病毒跨膜蛋白的囊膜蛋白但不是信号肽。结论:山羊痘病毒p32基因在大肠杆菌中获得高效表达,为山羊痘病毒的流行性调查、分子生物学研究以及以后有效防治本病提供了物质基础。Objective:To construct the prokaryotic expression vector PTYB11-gpvp32 for expression of goat pox(GPV) P32 protein and conduct preliminary analysis on favorable expression condition in E.coli BL21 cells.Method:First,GPVP32 sequence was analyzed using MEGA5.1,SOSUI and other bioinformatic software.Primer Primier 5.0 program was used to design primers in the upstream primer to add the restriction site of SpeⅠ and downstream primer to add restriction site of XhoⅠ.pSK-gpvp32 was used as the template for PCR amplification to obtain the gene fragment which was further cloned into PMD18-Simple-T to construct vector PMD18SimpleT-gpvp32,transformed into E.coli Top 10 competent cells,and screened by restriction enzyme digestion.After verification to the inserted sequence by sequencing,the gpvp32 gene fragment was cloned into the prokaryotic expression vector PTYB11 with restriction sites of SpeⅠ and XhoⅠ to prepare the prokaryotic expression plasmid PTYB11-gpvp32,transformed into E.coli BL21 and expressed upon induction by IPTG.Polyacrylamide gel electrophoresis(SDS-PAGE) and Western-blot were employed to analyze the expressed protein under different induction time.Result:The prokaryotic expression vector PTYB11-gpvp32 was successfully constructed and GPVP32 successfully expressed in E.coli BL21.Expression level of the recombinant protein reach approximately 28% of the total bacterial proteins.Evolutionary tree showed that the genetic relationship of goat pox and geographical differences and the result of evolution in different sheep,known by the SOSUI software such as a comprehensive analysis GPVP32 transmembrane protein of goat pox virus envelope protein but not the signal peptide.Conclusion:Goat poxvirus of p32 gene was successfully expressed in E.coli,which was prepared for Goat pox virus epidemic investigations,molecular biology research,and after the effective prevention and treatment of this disease.

关 键 词：山羊痘病毒 P32基因 原核表达 

分 类 号：S852.65[农业科学—基础兽医学]