[1]陈建飞,冯 力*,时洪艳,等.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,(11):856-860.
 CHEN Jian-fei,FENG Li*,SHI Hong-yan,et al.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,(11):856-860.
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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
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2007年11期
页码:
856-860
栏目:
病原生物学
出版日期:
2007-11-01

文章信息/Info

Title:
Genetic variation and prokaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain
文章编号:
1008-0589(2007)11-0856-05
作者:
陈建飞1冯 力1*时洪艳1孙东波1白兴华1佟有恩2
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
Author(s):
CHEN Jian-fei1 FENG Li1* SHI Hong-yan1 SUN Dong-bo1 BAI Xing-hua1 TONG You-en2
1. Division of Porcine Infectious Diseases, National Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150001, China;
2. Harbin Weike Biotechnology Development Company, Harbin 150001, China
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
Keywords:
porcine epidemic diarrhea virus N protein gene sequence identity the recombinant protein biologic activiey
分类号:
S852.659.6;Q786
文献标志码:
A
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1 326 bp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/03 N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54.4 Ku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。
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.

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备注/Memo

备注/Memo:
收稿日期:2007-01-29
作者简介:陈建飞(1974~),男,山东莱阳人,硕士,主要从事动物病毒分子生物学和分子免疫学研究.
*通讯作者:E-mail:fengli-h@163.com
更新日期/Last Update: 2011-01-17