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。
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 UL-38 fragment of pseudorabies virus（PRV） strain Ea was further subcloned into downstream of hexahistidine sequence prokaryotic expression vector pQE-82L,resulring in the prokaryotic expression plasmid pQE UL-38. After transformed into E. coli DH5a, expression of a fusion protein （6 × His-UL38d） with 40 000 molecular mass was observed in E. coli DHSu 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 ,18 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.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2009年第2期178-181,186,共5页
Chinese Journal of Veterinary Science
基金
国家自然科学基金资助项目(30400322)
