伪狂犬病病毒Ea株UL6基因的克隆、序列分析及表达

以伪狂犬病病毒Ea株基因组DNA为模板,通过PCR扩增UL6全长基因,将PCR产物克隆于pMD18-T载体,并采用双脱氧终止法进行序列测定.序列分析显示UL6全长1 938 bp,可编码646个氨基酸.将该基因克隆到插入原核表达载体pET28a的6×His下游,获得原核表达质粒pET28a-UL6,转化大肠埃希菌BL21,经IPTG诱导在大肠埃希菌中成功表达获得分子质量约70 ku的融合表达蛋白6×His-UL6,Western blot证实,表达的融合蛋白能与抗6×His的单克隆抗体发生特异性反应.根据测定的序列,设计一对能扩增UL6基因完整编码区的引物,PCR扩增UL6基因并将其插入真核表达载体pEGFP-C2中EGFP基因的3'端,获得与EGFP融合表达的真核表达质粒pEGFP-UL6,转染Hela细胞,通过激光共聚焦显微镜观察发现,转染48 h,融合蛋白EGFP-UL6主要定位在胞浆,为进一步研究伪狂犬病病毒Ea株UL6基因的结构和功能奠定了基础.

Cloning,Sequence Analysis and Expression of UL6 Gene of Pseudorabies virus Strain Ea

In this study,the UL6 gene of pseudorabies virus(PRV) strain Ea was amplified by PCR and cloned into pMDI8-T vector.The sequence of the gene was obtained by Sanger's sequencing technique.Sequence analysis showed that the UL6 gene is comprised of 1938 base pairs in length and encodes a 646 amino acid(aa).The full-length UL6 fragment of pseudorabies virus(PRV) strain Ea was further subcloned into downstream of hexahistidine sequence prokaryotic expression vector pET28a,resulting in the prokaryotic expression plasmid pET28aUL6.After transformed into E.coli BL21,expression of a fusion protein(6 His-UL6) with 70 ku molecular weight was observed in E.coli BL21 under induction with IPTG.The result of Western blot demonstrated that the fusion protein can be recognized by anti-6 His monoclonal antibody.According to result of sequencing UL6 gene was amplified and inserted into eurokaryotic expression vector pEGFP-C2,resulting in the expression plasmid pEGFP-UL6.After transfection into Hela cells,and detected at 48h post transfection,the fluorescence of the fusion protein EGFP-UL6 was observed on cell cytoplasm.The above results lay foundation for further studying structure and functions of UL6 gene of Pseudorabies virus(PRV) strain Ea.