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伪狂犬病毒Ea株UL38基因的克隆、序列分析及表达
  • ISSN号:1005-4545
  • 期刊名称:《中国兽医学报》
  • 时间:0
  • 分类:S852.659.6[农业科学—基础兽医学;农业科学—兽医学;农业科学—畜牧兽医]
  • 作者机构:[1]华中农业大学动物医学院动物病毒室,湖北武汉430070, [2]华中农业大学农业微生物学国家重点实验室,湖北武汉430070
  • 相关基金:国家自然科学基金资助项目(30400322)
中文摘要:

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UL-38基因全长1107bp,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40000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、48h通过激光共聚焦显微镜观察显示pEGFP-UL38,24h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48h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。

英文摘要:

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.

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期刊信息
  • 《中国兽医学报》
  • 北大核心期刊(2011版)
  • 主管单位:中华人民共和国教育部
  • 主办单位:吉林大学
  • 主编:王哲
  • 地址:吉林省长春市西安大路5333号
  • 邮编:130062
  • 邮箱:xbcjvs@163.com
  • 电话:0431-87836531 87836534
  • 国际标准刊号:ISSN:1005-4545
  • 国内统一刊号:ISSN:22-1234/R
  • 邮发代号:12-105
  • 获奖情况:
  • 第二届全国优秀科技期刊评比二等奖,连续多次被评为全军优秀医学期刊一等奖,全国高校优秀自然科学学报评比一等奖,中国期刊方阵“双百”期刊
  • 国内外数据库收录:
  • 美国化学文摘(网络版),英国农业与生物科学研究中心文摘,日本日本科学技术振兴机构数据库,中国中国科技核心期刊,中国北大核心期刊(2004版),中国北大核心期刊(2008版),中国北大核心期刊(2011版),中国北大核心期刊(2014版),中国北大核心期刊(2000版)
  • 被引量:18973