表达gpt和EGFP基因重组山羊痘病毒的构建和鉴定

在包含山羊痘病毒(Goatpox virus,GPV)疫苗株复制非必需区TK(Thymidine kinase)基因和背靠背启动子的质粒PtkPP的两个启动子下游插入EGFP(增强绿色荧光蛋白)基因,构建了质粒PtkPegfpP和PtkPPegfp,这两个重组质粒分别瞬时转染LT细胞,验证了启动子P11和P7.5能成功表达EGFP基因后,在PtkPP启动子P11的下游插入gpt(黄嘌呤鸟嘌呤磷酸转移酶)基因和EGFP基因,构建重组质粒PtkPgpt-egfpP,该重组质粒和GPV同源重组获得重组病毒rGPV-PtkPgpt-egfpP,利用MPA(霉芬酸)阻断核酸代谢途径筛选到稳定表达EGFP基因的重组GPV。为进一步开展GPV活载体疫苗的研究提供了技术平台。

Construction and identification of recombinant goatpox virus expressing gpt and EGFP gene

A goatpox virus transfer vector PtkPP, containing the fragments of the non-essential region was constructed with TK gene and the back to back promoter of goatpox virus vaccine strain. EGFP gene was inserted into downstream of the promoter 11 or promoter 7.5 to generate two individual transfer vector PtkPegfpP and PtkPPegfp. The two vectors were transfected into LT cells. The consistence expression of EGFP gene in the transfected cells demonstrated that the foreign gene could be expressed downstream either side of the back to back promoter. Fusion gene gpt and EGFP were then inserted into PtkPP downstream the late promoter 11, resulted in the generation of transfer plasmid PtkPgpt-egfpP. The recombinant virus was generated by transfection of PtkPgpt-egfpP into LT cells that had been infected with goatpox virus vaccine strain. This study supplied a tech- nical platform for developing goatpox virus live vector vaccine.