山羊痘病毒双向启动子序列的克隆与鉴定

摘要

In this study, a 56 bp sequence between ETF-1 and VUF-3 genes in Goatpox virus (GPV) genome was predicted to be a bidirectional promoter, and the sequence was synthesized and verified for bidirection of promoting activites by insert the GFP gene under control on either side of the 56 bp sequence, respectively. The recombinant transfer plasmids of pUC-TK12-Pb56(+) -EGFP and pUC-TK12-Pb56(-)-EGFP were constructed based on GPV TK gene as homologous arms and transfected into BHK cells preinfected with GPV, respectively, with the positive control of pUC-TK12-P7.5-EGFP and negative control of pUC-TK12-EGFP. The results demonstrated that the promoting activites of the 56 bp sequence on both directions were stronger than the P7.5 of Vaccinia virus evaluated by the assay of EGFP reporter gene expression. The findings of the present studies showed that the 56 bp bidirection promoter had a potential use in construction of the recombinant virus vaccines based on GPV.%为鉴定山羊痘病毒(GPV)基因组中的启动子序列,本研究预测GPV基因组中早期转录因子VETF-1和中期转录因子VITF-3基因序列之间56 bp的一段序列为双向启动子,并将其命名为Pb56.通过PCR技术将该启动子序列的两个方向(Pb56+,Pb56-)分别与绿色荧光蛋白(EGFP)报告基因融合,构建重组转移重组质粒,分别命名为pUC-TK12-Pb56(+)-EGFP和pUC-TK12-Pb56(-)-EGFP.用脂质体转染法将2个转移重组质粒以及阴性对照pUC-TK12-EGFP及阳性对照pUC-TK12-P7.5-EGFP分别转染GPV预感染的BHK细胞；采用EGFP报告基因的表达水平评估双向启动子的活性.结果表明该基因序列的两个方向均能启动EGFP的表达,初步证实了该基因序列为GPV的双向启动子；Pb56+和Pb56的转录活性均高于痘苗病毒的P7.5启动子.