猪圆环病毒2型河南分离株进化分析及ORF2基因的表达

根据GenBank中猪2型圆环病毒的核苷酸序列，设计两对引物，对来源于河南省不同地市的4株PCV2细胞培养物进行鉴定，并扩增出ORF2基因，克隆到pMD18－T载体上，获得重组质粒pMD18-T-ORF2，并对其进行测序，结果表明所克隆的ORF2基因与其它PCV2 ORF2基因核苷酸同源性在92.4%～99.6%之间，氨基酸同源性在90.6％～97.9％之间。同时采用PCR从重组质粒pMD18-T-ORF2中扩增出587bp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，经诱导表达了ORF2基因编码的结构蛋白，表达的重组蛋白为融合蛋白，分子量为40kD，经Western-Blot检测，重组蛋白可被PCV2阳性血清识别。

Phylogenetic analysis of Porcine Circovirus 2 isolated from Henan Province and Expression of the ORF2 Gene

Two pairs of primers were designed according to the published ORF2 gene sequence of PCV2, and ORF2 genes was amplified from PCV2 strain isolated from Henan province .The PCR products was cloned into pMD18-T easy vector and the recombinant plasmids named pMD18- ORF2 were obtained. The sequence of the cloned PCR product was analyzed and compared with other PCV2 isolate in GenBank. The results indicated that the homology of their nucleotide sequence was 92.4% to 99.6% ,and ORF2 gene displayed 90.6％to 97.9％identity based on amino acids with other PCV2 strains. Meanwhile, a fragment of ORF2 gene with a size of 587bp was amplified by PCR from pMD18-T-ORF2 of PCV2 and cloned into pET-32a vector .The recombinant plasmid was successfully expressed in Escherichia Coli BL21 by induction of IPTG. The molecular weight of fused recombinant protein was 40KD by SDS-PAGE and could be recognized by antiserum against PCV2 by Western-Blot.