大鲵虹彩病毒贵州分离株MCP基因的克隆与原核表达

根据Gen Bank中大鲵虹彩病毒主衣壳蛋白MCP(major capsid protein,MCP)基因序列(序列号:KF512820),设计一对特异性引物,以大鲵虹彩病毒贵州分离株基因组DNA为模板,PCR扩增大鲵虹彩病毒MCP基因并测序,与Gen Bank中大鲵虹彩病毒MCP基因进行比对,然后将其亚克隆到原核表达载体p ET-32a(+)中,转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导后进行Western blot分析。结果显示:PCR扩增出长度为1 392 bp的片段,与Gen Bank中大鲵虹彩病毒MCP基因核苷酸序列相似性为99.7%~99.9%,SDSPAGE电泳显示该重组蛋白的相对分子质量约为67×103。免疫原性检测结果表明,该重组蛋白可与兔抗大鲵虹彩病毒阳性血清特异性反应,具有免疫原性。

Cloning and prokaryotic expression of MCP gene of Giant salamander iridovirus from Guizhou strain

According to the MCP ( major capsid protein, MCP) gene sequence of Giant salamander iridovirus ( GenBank Accession No. KF512820)in GenBank, a pair of specific primer was designed for amplifying the specific fragments of MCP gene. The MCP gene was amplified from the Guizhou isolate of giant salamander iridovirus by PCR, and then was subcloned into the prokaryotic expression vector pET -32a ( +) . The recombinant plasmid pET -32a -MCP was transformed into E. coli BL21(DE3) and expressed under the induction of IPTG and SDS-PAGE. The results show that the MCP gene was 1392bp in length, the homology comparison between the MCP gene sequence and GenBank of MCP gene sequence showed 99. 7% ~99. 9% identities at nucleotide level. The result of SDS - PAGE showed the expressed protein was about 67 KD. Western-blot analysis indicated that the expressed protein could react with rabbit anti- GSIV serum, which had im-munological activity.