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  

Genetic variation and prokaryotic expression of N protein gene of porcine epidemic diarrhea virus CH/S strain

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] 

机构地区：[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

出　　处：《中国预防兽医学报》2007年第11期856-860,895,共6页Chinese Journal of Preventive Veterinary Medicine

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。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 anfi-PEDV hyperimmune serum in Westem blot.

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 

分 类 号：S852.659.6[农业科学—基础兽医学] Q786[农业科学—兽医学]