【作者】 金喜新；

【导师】 崔保安；

【作者基本信息】 河南农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本研究利用PCR技术对猪细小病毒HN1株NS1基因与VP2基因进行扩增,分别得到624bp和882bp的基因片段。将两个基因分别克隆到原核表达载体pGEX-4T-1中,构建了pGEX-NS1-HN1和pGEX-VP2-HN1原核表达载体,经酶切、PCR扩增和测序分析确证其正确插入到表达载体中,且阅读框正确。重组质粒转化至宿主菌BL21(DE3)中,用异丙基硫代-β-D-半乳糖苷(IPTG)进行诱导表达,用SDS-PAGE电泳、Western-blotting分析表达的蛋白。表达产物的分子质量分别约为52kDa和61kDa,与理论推测的蛋白分子质量一致;经Western-blotting证明pGEX-NS1-HN1重组蛋白可被感染猪细小病毒阳性血清所识别,然而pGEX-VP2-HN1重组蛋白可被猪细小病毒阳性血清所识别,表达蛋白可用于基因工程诊断抗原。 收集诱导表达的菌液,分离包涵体,尿素和Triton X-100分别洗涤包涵体,用尿素溶解包涵体,复性液稀释变性蛋白,缓冲液透析获取纯化蛋白。SDS-PAGE分析电泳结果表明,尿素和Triton X-100均可洗去部分杂蛋白。ELISA试验测定的结果显示,与复性前变性蛋白相比,纯化蛋白与PPV阳性血清反应的特异性提高30倍左右。可以批量生产基因工程诊断抗原来取代全病毒抗原,有望进一步研制针对NS1、VP2的单克隆抗体,为猪细小病毒病的诊断提供研究基础。 用纯化的猪细小病毒NS1蛋白和VP2蛋白作为包被抗原包被酶标板,建立了检测猪细小病毒抗体的间接ELISA方法,抗原包被浓度NS1蛋白和VP2蛋白分别为2.69μg/mL、2.29μg/mL,二抗(1:40000)37℃作用1h,底物溶液37℃显色5min。通过重复性试验、交叉试验、特异性试验、稳定性试验等试验结果表明该方法重复性好、特异性强、灵敏度高。为进一步组装成诊断试剂盒和鉴别诊断试剂盒奠定物质基础,为猪细小病毒的诊断与监测提供一种良好的技术手段。更多还原

【Abstract】 The NS1 gene and VP2 gene of porcine parvovirus (PPV) of HN1-strain were amplified by polymerase chain reaction (PCR). The amplified NS1 fragment of PPV was 624bp in length ; the amplified VP2 fragment of PPV was 882bp in length . The NS1 or VP2 fragments and expression vector pGEX-4T-l were digested by the same restriction endonucleases. These genes were ligated and transformed into Escherichia coli. The insert position, the size and the reading frame all were corrected by PCR, restriction digestion and the sequence analysis. The result showed that the prokaryotic expression vectors were constructed successfully. Then the recombinants were transformed into BL21 (DE3) for gene expression with IPTG inducing. The expressed proteins were measured by SDS-PAGE and western-blotting. The results showed that the NS1 gene and VP2 gene can express successfully in E.coli. The western-blotting results indicated that the expressed proteins could be recognized by the PPV positive serum. These results showed that expressed proteins can be used in genetic engineering diagnostic antigen .The inclusion bodies were obtained by sonication of the bacterial cells, from which the purified activative expressed recombinant NS1 proteins and VP2 proteins were got by urea and Triton X-100 washing and by renaturing, refolding and dialyzing. The results of SDS-PAGE show that washing by urea and Triton X-100 can decrease the amounts of bacteria proteins in the inclusion body. The reactivity of purified protein was increased 30 times in ELISA experiments comparing with the routine antigen. The diagnostic antigen that is substitution of the whole viral antigen may be produced in batches. It is hopeful to develop the monoclonal antibody against NS1、VP2 protein further. Offered advanced technology for diagnosis of the disease of porcine parvovirus.Based on the purified recombinant proteins, an indirect ELISA method for detection of PPV antibodies was developed.The concentration of coating antigen of NS1 is 2.69μg/ml and VP2 is 2.29μg/ml, HRP labeled anti-porcine IgG (1: 40000) being incubated at 37□ for 1h. The ELISA assay was confirmed to have a good reiterativity, specificity and sensitivity by repeated test, crossing test and blocking更多还原