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猪流行性腹泻病毒CH/S株N蛋白基因的遗传变异及其原核表达
引用本文:陈建飞,冯力,时洪艳,孙东波,白兴华,佟有恩.猪流行性腹泻病毒CH/S株N蛋白基因的遗传变异及其原核表达[J].中国预防兽医学报,2007,29(11):856-860,895.
作者姓名:陈建飞  冯力  时洪艳  孙东波  白兴华  佟有恩
作者单位:1. 中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室/猪传染病研究室,黑龙江,哈尔滨,150001
2. 哈尔滨维科生物技术开发公司,黑龙江,哈尔滨,150001
摘    要:应用RT-PCR和nested-PCR方法扩增得到含猪流行性腹泻病毒CH/S株N蛋白基因的目的片段,并将其进行了克隆、序列测定及分析。CH/S株N蛋白基因含有一个长1326bp的ORF,编码由441个氨基酸残基组成的多肽,未发现碱基的插入和缺失。CH/S与CV777、Chinju99、JS-2004-2和LJB/03N蛋白基因ORF的序列同源性分别为97.1%、96.8%、96.7%和96.5%;推导的氨基酸序列的同源性分别为97.7%、97.1%、97.1%和96.8%。以阳性质粒为模板,用分别含有BamHⅠ和XhoⅠ酶切位点的上、下游引物扩增得到ORF,其PCR产物经BamHⅠ和XhoⅠ双酶切后定向克隆到pET-30a载体,构建的重组质粒命名为pET-30a-PN;将pET-30a-PN转化到大肠杆菌BL21(DE3)中,在IPTG诱导下进行表达;SDS-PAGE结果表明表达出与预期大小相符的约54.4Ku的重组蛋白,重组蛋白以包涵体形式存在;薄层扫描结果表明表达产物占菌体总蛋白的30.5%;Western blot分析表明表达的重组蛋白能与抗PEDV高免血清反应,说明该重组蛋白具有免疫学活性。

关 键 词:猪流行性腹泻病毒  N蛋白基因  序列同源性  重组蛋白  免疫学活性
文章编号:1008-0589(2007)11-0856-05
修稿时间:2007-01-29

Genetic variation and prokaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain
CHEN Jian-fei,FENG Li,SHI Hong-yan,SUN Dong-bo,BAI Xing-hua,TONG You-en.Genetic variation and prokaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain[J].Chinese Journal of Preventive Veterinary Medicine,2007,29(11):856-860,895.
Authors:CHEN Jian-fei  FENG Li  SHI Hong-yan  SUN Dong-bo  BAI Xing-hua  TONG You-en
Institution:1. Division of Porcine Infectious Diseases, National Key Laboratory of Veterinary Bioteehnology, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China; 2. Harbin Weike Biotechnology Development Company, Harbin 150001, China
Abstract:The N protein gene of porcine epidemic diarrhea virus CH/S strain was amplified by RT-PCR,and sequence compared with four PEDV reference strains.The ORF of N protein gene of CH/S strain comprised 1 326 nt encoding a polypeptide of 441 amino acids residues.There was no deletion and insertion in the coding region.The ORF shared 97.1%,96.8 %,96.7 % and 96.5% nucleotide sequence homology and 97.7%,97.1%,97.1% and 96.8% amino acid homology with that of the CV777, Chinju99,JS-2004-2 and LJB/03,respectively.The N gene ORF was then subcloned into pET-30a vector and the recombinant plasmid was transformed into E.coli BL21(DE3)and induced with IPTG.The protein expression was determined by SDS-PAGE. The expressed protein had a molecular weight of 54.4 Ku that existed as inclusion body.Thin-layer scanning showed that the expression product accounted for 30.5% of the total bacterial proteins.The recombinant protein possessed native biological activity and could react with anti-PEDV hyperimmune serum in Western blot.
Keywords:porcine epidemic diarrhea virus  N protein gene  sequence identity  the recombinant protein  biologic activiey
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