表达GFP基因的马立克氏病病毒(MDV)转移载体的构建及初步应用

利用Primer primer 5.0软件设计两条引物，将loxP位点分别引入正、反向引物，以质粒载体pIRES-gpt为模板，扩增获得LoxP-CMV-gpt-IRES-LoxP基因表达盒(约2.9kb)，将其克隆入质粒pBUS10 (含有MDV US10区) 的BalⅠ位点，获得重组质粒 pUS-gptIRES(L)；对重组质粒pUS-gptIRES(L)进行测序分析，发现两同向的loxP位点正确插入到pUS-gptIRES(L)中；将含有完整的GFP基因以及poly A尾的基因片断，连入pUS-gptIRES(L)中，把gpt基因替换掉，从而获得重组质粒pUS-GFPIRES(L)。将转移载体pUS-GFPIRES(L)瞬时转染CHO细胞，可以观察到绿色荧光，说明GFP基因获得有效表达；将pUS-GFPIRES(L) 转染已感染MDV CVI988株的CEF细胞，可以筛选到表达绿色荧光蛋白的重组病毒；通过测定重组病毒在细胞上的生长曲线，发现重组病毒与亲本毒生长曲线相吻合。本研究为进一步探讨MDV在体内的特性奠定基础，同时，为Cre/LoxP重组酶系统在MDV中的应用奠定基础。

Construction of Transferring Vector of Marek's Disease Virus Expressing GFP gene and its Primary Application

The expressing cassette, LoxP-CMV-gpt-IRES-LoxP( about 2.9kb), was amplified by PCR from a plasmid, pIRES-gpt, by use of the primers , which contained the loxP sites in 5' terminals, respectively. The loxP sites were designed into primers by the software of Primer primer 5.0. Then the cassette was cloned into the site of BalI in pBUS10 to obtain pUS-gptIRES(L). The sequencing analysis for pUS-gptIRES(L) indicated that two loxP sites with the same direction were correctly inserted into pUS-gptIRES(L).The gpt gene in pUS-gptIRES(L) was replaced by a fragment including the full length GFP gene as well as SV40 poly A sequence to get pUS-GFPIRES(L). pUS-GFPIRES(L) was transiently transfected into CHO cell lines, and then the green fluorescence could be seen, the results showed that GFP gene could be expressed correctly. Moreover, pUS-GFPIRES(L) was transfected into the CEF infected MDV CVI988 strain and recombinant virus was selected by the green fluorescence. The growth curve of virus showed the characteristic of recombinant virus was the same as that of CVI988 in vitro. These results give the basis for further studying the characteristic of MDV in vivo and the application of the Cre/LoxP system to MDV genome.