猪细小病毒NS1蛋白的截短表达与间接ELISA诊断方法的建立

Development of an indirect ELISA for detecting antibodies to porcine parvovirus using recombinant truncated NS1 protein expressed in E.coli

【目的】建立一种快速检测猪细小病毒(Porcine parvovirus,PPV)野毒抗体的方法。【方法】参照已发表的PPV基因组(AY502114.1)序列设计合成特异性引物,利用PCR扩增PPV NS1基因主要抗原区域,将目的片段定向克隆至表达载体pET30a(+),转化BL21(DE3)表达菌,用IPTG诱导表达重组蛋白。以该蛋白作为诊断抗原,经过对间接ELISA条件的优化,建立检测PPV抗体的NS1-ELISA诊断方法,对该方法的特异性、敏感性、重复性进行检测。用建立的PPV NS1-ELISA诊断方法和血凝抑制试验(HI)同时对临床送检的306份血清样品进行检测,比较二者的符合率。【结果】成功扩增了870bp的NS1基因部分片段,构建了pET30a-NS1原核表达载体,获得了以包涵体形式表达的重组蛋白。该重组蛋白具有良好的抗原性和特异性。以纯化蛋白作为诊断抗原,建立了检测猪细小病毒抗体的NS1-ELISA诊断方法。该方法与猪圆环病毒2型(PCV-2)、猪伪狂犬病病毒(PRV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪乙脑病毒(JEV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)、猪瘟病毒(CS-FV)等7种常见猪病病毒的阳性血清均不发生反应;该方法检测敏感性为1∶12 800;批内重复性试验和批间重复性试验中样品的变异系数分别小于5%和10%。PPV NS1-ELISA检测方法与HI的符合率为96.2%。【结论】制备了NS1重组蛋白,该蛋白具有良好的免疫原性,以其作为包被抗原建立的PPV NS1-ELISA诊断方法具有良好的重复性、敏感性和特异性,为PPV的快速诊断和流行病学调查等提供了一种快速、简便的血清学诊断方法。

【Objective】 The study developed a method for quick detection of porcine parvovirus antibody.【Method】 NS1 gene major antigen region was amplified by RT-PCR technique with specific primers based on published PPV genome sequence（AY502114.1）.Then the target fragment was directly cloned into pET30a（＋） vector.The recombinant plasmid was transformed into E.coli BL21（DE3）.The recombinant proteins were expressed after induced by IPTG.Using the purified recombinant NS1 protein as coating antigen,an indirect NS1-ELISA was developed to detect antibody to PPV,and to test the specificity,sensitivity and repeatability of the method.With the established PPV antibody NS1-ELISA diagnosis method and hemagglutination inhibition test（HI）,306 serum samples were detected,and its coincidence rate was compared.【Result】 870 bp NS1 gene fragments were successfully amplified,prokaryotic expression vector pET30a-NS1 was constructed,and a recombinant protein in inclusion body forms was obtained.After denaturation,purification and renaturation,the purified recombinant protein retained better antigenicity and specificity.An indirect ELISA was successfully developed using the purified recombinant NS1 protein as coating antigen.The method showed no cross-reaction with the positive sera of other seven swine diseases（PCV-2,PRV,PRRSV,JEV,PEDV,TGEV,CSFV）.The sensitivity of the method was 1∶12 800.Coefficient of variability percent（C.V%） of intro-batch and inter-batch duplicative tests was less than 5% and 10%,respectively.Compared with the HI,the concordance was 96.2%.【Conclusion】 Recombinant NS1 protein was prepared.This recombinant protein has a good immunogenicity.The NS1-ELISA has good repeatability,sensitivity and specificity,which could be used as a simple and rapid serology detection method to monitor anti-PPV antibody and epidemiologic survey of PPV.