轮状病毒VP4全基因的序列分析及其在原核系统中的初步表达  

Sequence analysis and prokaryotic expression of full length VP4 gene of rotavirus group A

作　　者：霍云雯[1] 钱渊[1] 李国华[1] 肖玮[1] 

机构地区：[1]首都儿科研究所北京市感染与免疫中心实验室,100020

出　　处：《中华儿科杂志》1999年第11期680-683,共4页Chinese Journal of Pediatrics

基　　金：国家自然科学基金;北京市自然科学基金

摘　　要：目的　研究轮状病毒（ＲＶ） 我国地方株ＶＰ４ 基因特点，并获得高效表达的ＶＰ４。　方法采用ＲＴＰＣＲ 技术扩增ＲＶＰ１Ａ 型我国地方株ＣＲ１８８ 的完整ＶＰ４ 基因，克隆到原核表达质粒ｐＥＴ２１ａ（＋ ）中：（１）用末端终止法进行核苷酸序列测定，并与人（ＨＲＶ）标准株作比较分析。（２） 重组质粒转化原核表达宿主菌Ｅ－ｃｏｌｉ ＢＬ２１（ＤＥ３） ，异丙硫半乳糖甙（ＩＰＴＧ） 诱导表达，经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（ＳＤＳＰＡＧＥ） 和Ｗｅｓｔｅｒｎ ｂｌｏｔ 对其表达产物进行鉴定。结果　（１）ＣＲ１８８ ＶＰ４ 基因全长２３５９ｂｐ，含编码７７５ 个氨基酸的单一的开放读码框架（ＯＲＦ） ，与相同血清型标准株之间氨基酸序列具有高度同源性（９４ ％ ～９８％ ），与不同血清型标准株间相比较，则同源性较低（６４ ％ ～７８％ ） 。（２） 重组ＶＰ４表达量在诱导２～３ 小时时最高，占细胞总蛋白的１１ ％ ；表达蛋白与小鼠抗Ａ组ＲＶＶＰ４ Ｐ１Ａ型特异性单克隆抗体（ 单抗） 发生反应。结论　序列分析预测血清型的结果与临床标本的实验室诊断一致，ＣＲ１８８ ＶＰ４ 为Ｐ１Ａ型；ＶＰ４ 全基因在原核系统中获得表达，表达的重组ＶＰ４ 抗原可被型特?Objective To characterize VP4 gene of human rotavirus field strain in China and obtain highly expressed VP4 protein Methods Full length VP4 gene of human rotavirus CR188 strain was amplified by RT PCR and cloned into prokaryotic expression plasmid pET21a(+): (1) The VP4 gene was sequenced with terminal termination method and compared with previously published strains (2) The recombinant plasmid was transformed into E coli BL21 (DE3) and induced by isopropyl thiogalactoside (IPTG) The expressed protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blot Results (1) The cloned VP4 gene was 2359 bp in length and had a long open reading frame (ORF) coding for 775 amino acids Comparative analysis of the deduced amino acid sequences of VP4 indicates a high degree of homology among the same serotypes (94% 98%) and a low degree of homology among the various serotypes (64% 78%) (2) The maximal quantity of expressed protein was harvested at the hours 2 3 following induction and showed 11 % of expression level in the whole cell protein Monoclonal antibodies against group A rotavirus VP4 could specifically recognize the expressed protein epitopes in Western blot assay Conclusion Sequence analysis and laboratory diagnosis using clinical sample indicated consistently that the serotype of CR188 was P1A; The VP4 gene was highly expressed in prokaryotic system and with the same antigenicity of natural VP4

关 键 词：轮状病毒属 基因 结构 病毒 基因表达 序列分析 

分 类 号：R373.2[医药卫生—病原生物学]