猪圆环病毒Ⅱ型河南地方株ORF4的克隆及原核表达
Journal of Henan Institute of Science and Technology (Natural Sciences Edition)(2014)
Abstract
根据GenBank数据库中猪圆环病毒Ⅱ型的基因组序列设计引物,采用PCR技术从病料基因组DNA中扩增出PCVⅡ河南地方株ORF4基因,全长180 bp,编码59个氨基酸.将该基因克隆至载体pGEX-4T-3中形成pGEX-4T-3-ORF4表达载体.经PCR、酶切和测序鉴定后,转化表达菌株BL21(DE3)诱导表达.SDS-PAGE结果显示:ORF4能够在大肠杆菌中表达,产物的分子量约为32 kD,且以包涵体形式存在.Western Blot检测结果显示,纯化后的ORF4蛋白能够与鼠抗6×His标签单克隆抗体发生特异性反应,为进一步研究PCVⅡORF4蛋白的特性与功能奠定了基础.
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Key words
Porcine circovirus typeⅡ,ORF4 gene,prokaryotic expression
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