马立克氏病病毒CV1988株pp24基因的克隆与表达

Cloning and Expression of pp24 Gene of CV1988 Strain of Marek's Disease Virus

根据马立克氏病病毒 (MDV)国际参考强毒GA株 pp2 4基因序列 ,设计和合成了一对引物 ,其两端分别含SalI和EcoRI的酶切位点。以CV1988株基因组DNA为模板 ,通过PCR技术 ,扩增其pp2 4基因。PCR产物经纯化后 ,按正确的阅读框架定向克隆到表达性载体pGEX_6P_1中谷胱甘肽转移酶 (GST)基因的下游 ,并用序列测定验证。将重组质粒转化的大肠杆菌BL2 1,在 1.0mMIPTG和 37℃条件下诱导 ,GST_pp 2 4基因获得了表达。诱导菌体的裂解物经 12 %的SDS聚丙烯凝胶电泳 (SDS_PAGE)和Westernblot试验验证 ,得到大小为 4 3kD的融合蛋白 ,与预期大小一致。将该融合蛋白的凝胶带切下研碎后免疫小鼠三次后 ,其血清与MDV感染的鸡胚成纤维细胞 (CEF)

Marek's Disease Virus(MDV)pp24 gene was amplified from CV1988 strain by Polymerase Chain Reaction(PCR).PCR product was cloned to the downstream of GST gene according to the right Open Reading Frame(ORF) in pGEX_6P_1 vector,and E.coli BL21 was transformed by the recombinant plasmid for expression.The expressed product was identified and pruified through SDS_PAGE,and a 43KD fusion protein with GST was found.The specific band was excised from the gel and injected into mice.The antiserum was collected from the immunized mice and used for Indirect Fluorescent Assay(IFA) with Chicken Embryonic Fibroblast(CEF) infected by MDV.The positive staining were found in the MDV plaques and localized on the cytoplasm of the infected cell.The result showed that the in vitro expressed protein of pp24 genes has some effective epitopes of MDV.