猪瘟病毒NS3基因克隆、原核表达及间接ELISA方法初步建立  

Cloning,Prokaryotic Expression of CSFV NS3 Gene,and Preliminary Establishment of an Indirect ELISA for Serum Antibody Detection

作　　者：蒋大良[1] 余兴龙[1] 李润成[1] 葛猛[1] 罗维[1] 颜爱[1] 李杰[1] 刘春红[1] 涂长春[2] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128 [2]军事医学科学院军事兽医研究所,吉林长春130062

出　　处：《微生物学通报》2010年第1期78-84,共7页Microbiology China

基　　金：国家科技支撑计划项目(No2006BAD06A00);湖南省科技重大专项(No2007FJ1003)

摘　　要：采用PCR方法从携带猪瘟病毒兔化弱毒(Hog cholera lapinized virus,HCLV)全长基因组cDNA的质粒pPOHCLV中扩增到长度为2000bp左右NS3基因序列,并将其克隆至原核表达载体pET-32a(+),构建成重组原核表达载体pETNS3。将pETNS3在大肠杆菌Rosetta(DE3)中进行优化表达,SDS-PAGE分析重组蛋白NS3主要以包涵体形式表达,分子大小约95kD。Western Blotting分析表明重组蛋白NS3具有免疫原性。采用Ni+亲和层析方法纯化得到重组蛋白NS3(90%)。以纯化的重组蛋白NS3为抗原初步建立了检测CSFVNS3抗体的间接ELISA方法,检测221份不同猪群和年龄猪的血清样品。检测结果与IDEXX公司CSFV-Ab检测试剂盒检测结果进行对比,阳性符合率为83.33%,阴性符合率为89.38%,总符合率为86.43%。30份存在差异的血清样品用间接免疫荧光法(Indirect immunofluorescence assay,IFA)进行检测,结果显示IFA检测结果与NS3间接ELISA和IDEXX公司CSFV-Ab检测试剂盒符合率分别为56.67%和43.33%。NS3 gene fragment （about 2000 bp） was amplified by PCR from plasmid ofpPOHCLV containing Hog Cholera Lapinized Virus （HCLV） cDNA, and cloned into the prokaryotic expression vector pET-32a （＋） to obtain the recombinant prokaryotic expression vector pETNS3. The pETNS3 was transformed into E.coli Rosetta （DE3） and expressed optimally. The recombinant protein NS3 about 95 kD was detected by SDS-PAGE and expressed mainly in the form of inclusion bodies. The result of Western blotting showed re-combinant protein NS3 has immunogenicity. The nickel affinity chromatography was employed to purify the target protein, and purified recombinant protein NS3 （90%） was obtained. An indirect ELISA was initially established to detect antibody against CSFV NS3 protein, and 221 sera samples of pig from different herds and ages were tested. The result was compared with CSFV-Ab test kit of IDEXX, and the result showed： the positive coincidence rate was 83.33%, the negative coincidence rate 89.38%, and the total coincidence rate 86.34%. 30 serum samples showed inconsistent, and were detected by Indirect Immunofluorescence Assay （IFA）. The results showed： compared with IFA the accuracy rate of result detected by NS3 indirect ELISA and CSFV-Ab test kit of IDEXX was 56.67% and 43.33% respectively.

关 键 词：CSF V NS3 基因克隆 原核表达 蛋白纯化 免疫印迹 间接ELISA IFA 

分 类 号：Q78[生物学—分子生物学] TS207.3[轻工技术与工程—食品科学]