【摘要】 以伪狂犬病毒Ea株基因组DNA为模板,通过PCR扩增UL38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UL38基因全长1 107 bp,可编码369个氨基酸。将该基因克隆到原核表达载体pQE-82L的6×His下游,获得原核表达质粒pQE-UL38,IPTG诱导在大肠杆菌中成功表达并获得相对分子质量约40 000的融合表达蛋白6×His-UL38,Western blot试验证实表达的融合蛋白能与抗6×His的单抗发生特异性反应。进一步将UL38基因插入真核表达载体pEGFP-N1中EGFP基因的5′端,获得与EGFP融合表达的真核表达质粒pEGFP-UL38,转染Hela细胞,24、48 h通过激光共聚焦显微镜观察显示pEGFP-UL38,24 h荧光主要分布在胞浆,有部分分布在核内,但随时间延长,荧光逐渐向细胞核中转移,在转染后48 h几乎完全定位于核内。但对照载体pEGFP-N1转染细胞的荧光一直呈弥散型分布于整个细胞。更多还原

【Abstract】 In this study,the U38 gene of pseudorabies virus(PRV) strain Ea was amplified by PCR and cloned into pMDI8-T vector,using the genome of PRV strain Ea as templates.The sequence of the gene was obtained by Sanger’s sequencing technique.Sequence analysis showed that the UL38 gene is comprised of 1 107 base pairs in length and encodes a 369 amino acid(aa) protein.The full-length UL38 fragment of pseudorabies virus(PRV) strain Ea was further subcloned into downstream of hexahistidine sequence prokaryotic expression vector pQE-82L,resulting in the prokaryotic expression plasmid pQE-UL38.After transformed into E.coli DH5α,expression of a fusion protein(6×His-UL38d) with 40 000 molecular mass was observed in E.coli DH5α under induction with IPTG.The result of Western blot demonstrated that the fusion protein can berecognized by anti-6×His monoclonal antibody.Then UL38 gene was inserted into eurokaryotic expression vector pEGFP-N1,resulting in the expression plasmid pEGFP-UL38.After transfection into Hela cells,and detected at 24 h and 48 h post transfection.The results showed that the fluorescence mainly located on cytoplasm,partly located on cell nucleolus at 24 h,but mainly on cell nucleolus at 48 h.However the fluorescence in pEGFP-N1-transfected cells was always uniformly distributed,localizing throughout the cytoplasm of the cell.更多还原