猪圆环病毒2型(PCV2)河南分离株进化分析及ORF2基因的表达  

Phylogenetic Analysis of Porcine circovirus 2(PVC2) Isolated from Henan Province and Expression of the ORF2 Gene in Escherichia coli

作　　者：陈陆[1] 杨霞[1] 王江辉[1] 常洪涛[1] 董加才[1] 刘矿[1] 刘红英[1] 王川庆[1] 

机构地区：[1]河南农业大学牧医工程学院,郑州450002

出　　处：《农业生物技术学报》2009年第4期561-566,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2007AA100606);国家支撑计划(No.2006BAK02A21-3)资助

摘　　要：根据GenBank中猪圆环病毒2型(Porcine circovirus2,PCV2)的核苷酸序列设计2对引物,使用扩增全基因的引物对来源于河南省焦作、开封和滑县的3株PCV2型JZ株、KF株和HX株的ORF2基因进行扩增,扩增片段克隆到pMD18T上,获得重组质粒pMD18-T-ORF2,并对其进行测序,测序结果登录在GenBank上,登录号分别为EF028202、EF064149和EF467928。序列分析表明,PCV2河南分离株ORF2基因与其它PCV2ORF2基因核苷酸序列同源性为92.4%～99.6%,氨基酸序列同源性为90.6%～97.9%,进化分析表明,河南分离株处于同一分支,与欧洲株亲缘关系较近。应用另一对引物从重组质粒pMD18-T-ORF2中PCR扩增出587bp不包含核定位信号序列的ORF2基因,克隆到表达载体pET-32a,成功构建了重组质粒pET-32a-ORF2,经IPTG诱导表达了ORF2基因编码的结构蛋白,经SDS-PAGE和Western blotting检测,表达的重组蛋白分子量约为40kD,可被PCV2阳性血清识别,说明PCV2ORF2基因得到成功表达。Two pairs of primers were designed according to the published ORF2 gene sequence of Porcine circovirus 2（PCV2）, and ORF2 genes was amplified from PCV2 strain JZ, KF and HX isolated from Henan Province. The PCR products were cloned into pMD 18 T easy vector and the recombinant plasmids named pMD 18-T-ORF2 were obtained and sequenced. The sequences had been submitted to GenBank databases and the accession numbers were EF028202, EF064149 and EF467928, respectively. The sequences were analyzed and compared. The results indicated that the ORF2 gene ofPCV2 Henan isolates had similarities ranging from 92.4% to 99.6% at nucleotides level and 90.6% to 97.9% at amino acids level with other PCV2 isolates in GenBank. 587 bp truncated ORF2 gene without nuclear localization signal （NLS） sequence was amplified by PCR from pMD18-T-ORF2 of PCV2 JZ strain and subcloned into pET-32a vector. The recombinant plasmid pET32a-ORF2 was successfully expressed in Eschedchia coli BL21 by induction of IPTG. SDS-PAGE and Western blotting analysis indicated that the fusion protein was approximately 40 kD in molecular weight and could be recognized by antiserum against PCV2.

关 键 词：猪圆环病毒2型(PVC2) ORF2基因 序列分析 表达 

分 类 号：S188[农业科学—农业基础科学]