猪传染性胃肠炎病毒(TGEV)福建分离株核衣壳蛋白基因(N)的克隆与表达分析  

Cloning and Expression Analysis of Nucleocapsid Protein-encoding Gene(N) from Porcine Transmissible gastroenteritis virus(TGEV) Fujian Strain

作　　者：王劭[1,2] 朱小丽[1] 朱晓琳[1,3] 陈少莺[1,2] 林锋强[1,2] 陈仕龙[1,2] 程晓霞[1,2] 李兆龙[1,2] 江斌[1,2] 

机构地区：[1]福建省农业科学院畜牧兽医研究所 [2]福建省畜禽疫病防治工程技术研究中心,福州350013 [3]福建农林大学动物科学学院,福州350002

出　　处：《农业生物技术学报》2013年第1期112-119,共8页Journal of Agricultural Biotechnology

基　　金：福建省农科院创新团队项目(STIF-Y02);福建省公益类科研院所专项(No.2010R1025-1)

摘　　要：猪传染性胃肠炎(swine transmissible gastroenteritis,TGE)是一种高度接触性肠道传染病,是我国法定检疫的疫病。为研究福建省猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)核衣壳蛋白基因(N)的分子特征及其原核表达产物的抗原性,参照GenBank中的TGEV全基因序列,设计合成一对扩增TGEV福建(FJ)分离株N基因的特异性引物,进行克隆和序列分析,结果表明,TGEV-FJ株N基因全长1149bp,编码382个氨基酸(GenBank登录号JQ700302),与国内外23株TGEVN基因的核苷酸同源性,氨基酸同源性进行比较分析,N基因核苷酸的同源性为95.4%～99.8%,氨基酸同源性为96.1%～100%。TGEV-FJ株N基因克隆至原核表达载体pET-32a中,并转化到大肠杆菌(Escherichia coli)Rosetta(DE3)中进行表达。SDS-PAGE分析表明,重组蛋白约为68kD,与预期分子量一致;Western blot分析表明,重组蛋白能与抗TGEV阳性血清反应出现特异性条带;以纯化的重组TGEVN蛋白为包被抗原建立的间接ELISA抗体检测方法具有良好的特异性,送检20份猪(Sus scrofa)TGEV阳性血清样本中检出16份阳性结果、10份阴性血清样本中检出9份阴性结果。利用纯化的TGEV重组N蛋白免疫Balb/c小鼠,杂交瘤技术制备单克隆抗体,获得了1株特异性好并能稳定分泌抗TGEVN蛋白的单克隆抗体的杂交瘤细胞株(命名为1-27),间接免疫荧光试验证明,该单抗能特异性识别TGEV全病毒抗原。TGEVN蛋白的单克隆抗体制备为建立TGEV免疫学检测方法提供了基础资料。Transmissible gastroenteritis （TGE） is a coronavirus which causes enteric disease in swine of all ages. To investigate the sequence characteristic of the nucleocapsid protein gene （N） and its prokaryotic expression product of the porcine Transmissible gostroenteritis virus in Fujian Province （TGEV-FJ）, the N gene was amplified by RT-PCR with primers designed according to TGEV strains in GenBank. The sequence analysis demonsrated that the N gene of TGEV-FJ strain was 1 149 bp encoding a protein of 382 amino acids （GenBank accession No. JQ700302）, which shared high degree of identity for nucleotide and amino acid sequences with TGEV reference strains （95.4%to99.8% and 96.1%to100%, respectively）. The N gene was cloned into pET-32a （＋）-C vector for expression in E.coil Rosetta （DE3） after IPTG induction. SDS-PAGE analysis showed that the N product was 68 kD. Western blot demonstrated that the expressed recombinant protein was recognized specifically with porcine anti TGEV-FJ serum. An indirect ELISA coated with the purified recombinant nucleocapsid protein showed specificity for the detection of antibody against TGEV, 16 out of 20 sera from piglets （Sus scrofa） infected with TGEV were positive, and 9 out of 10 healthy sera were negative. Balb/c mice were immunized with the purified expression product of N protein. One hybridoma cell line （named 1-27）stably secreting McAbs against pET-32a-N was obtained, and the McAbs had strong reaction with TGEV antigen by indirect immunofluorescence assay （IFA）. The McAbs prepared in this study can be served as a useful tool for developing immunological diagnostic techniques for TGEV infection.

关 键 词：猪 传染性胃肠炎病毒(TGEV) TGEV-福建(Fujian)分离株 核衣壳蛋白 N基因 原核表达 

分 类 号：S852.659.6[农业科学—基础兽医学]