兰州地方分离PCV毒株ORF2阅读框的测序分析与原核表达鉴定  

Sequence Analysis and Prokaryotic Expression Identification of ORF2 Gene in Porcine Circovirus Isolated from Lanzhou

作　　者：傅昱[1] 郝晓芳[2] 卢曾军[2] 孙普[2] 胡永浩[1] 刘在新[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所,甘肃兰州730070

出　　处：《湖南农业科学》2010年第4期23-27,共5页Hunan Agricultural Sciences

基　　金：国家支撑计划(2006BAD06A12);国家"973"项目(2005CB523201)

摘　　要：根据GenBank中发表的PCV1和PCV2的ORF2基因序列,设计合成了一对共用引物,用PCR的方法从兰州本地病料中分离出毒株,通过PK-15的增殖,扩增出ORF2序列,测序分析后发现分离毒株序列与PCV1的ORF2序列同源性达到99.1%～99.4%。根据GenBank中PCV2的ORF2序列对目的序列进行了改造、优化和合成后,目的序列和pET-32a载体连接,转化BL21(DE3)细胞后,挑取阳性克隆。对鉴定后的融合质粒用IPTG诱导,裂解液经过SDS-PAGE分析、纯化操作和Western-Bloting分析,表明改造合成ORF2序列在大肠杆菌中得到了表达,并能被PCV2阳性血清所识别。According to the nucleotide sequences of ORF2 in porcine circovirus type 1（PCV1） and type 2（PCV2） published in GeneBank,a pair of common primers to the ORF2 gene in PCV1 and PCV2 were synthesized.Using PCR method to isolate virus strain from Lanzhou local sample,and then ORF2 gene was amplified through proliferation of PK-15 cell.Through sequence analysis and homology comparison,the results indicated that sequence of ORF2 in isolated virus strain shared 99.1%～99.4% of homology with ORF2 in PCV1.The purpose sequence has reformed,optimized and synthesized based on the gene sequence of ORF2 in PCV2 in GeneBank,and then it was subcloned into prokaryotic expression vector pET-32a.The resultant ORF2-pET 32a was transformed into E.coli BL21（DE3） cell and induced by IPTG.The results of SDS-PAGE,purification and Western-Bloting analysis indicated that the reconstructed ORF2 gene sequence had been expressed in E.coli,and it could be recognized by positive serum of PCV2.

关 键 词：PCV 分离鉴定 ORF2基因优化 克隆 表达 

分 类 号：Q786[生物学—分子生物学]