猪繁殖与呼吸综合征病毒重组M蛋白间接ELISA抗体检测方法的建立及应用

为建立一种敏感、特异、快速、高通量的猪繁殖与呼吸综合征病毒(PRRSV)抗体血清学检测方法,本研究利用原核表达技术表达了PRRSV M蛋白,将纯化后的重组M蛋白作为包被抗原建立了检测PRRSV抗体的间接ELISA方法。参照已发表的PRRSV基因组M基因序列,设计合成1对特异性引物,RT-PCR扩增了长约435 bp的M基因片段,将目的片段亚克隆至pET32a(+)表达载体中,经IPTG诱导获得了以包涵体形式表达的重组M蛋白,重组蛋白纯化后,免疫印迹检测结果表明具有良好的抗原性和特异性。以重组M纯化蛋白为包被抗原,经间接ELISA反应条件的优化,建立了检测PRRSV抗体的间接ELISA方法,该方法检测猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病病毒(PRV)、猪传染性胃肠炎病毒(TGEV)、猪圆环病毒2型(PCV2)其他6种常见猪病病原的阳性血清均为阴性;该方法批内与批间重复性试验的变异系数分别小于5%和10%;该方法与商品化ELISA试剂盒的符合率为95.3%。本研究建立的M-ELISA检测方法将为猪群免疫PRRS疫苗后抗体水平监测及PRRSV野毒感染的快速诊断与流行病学调查等提供了一种简便易行、快速、高通量的血清学抗体检测方法。

Establishment and Application of an Indirect ELISA with Recombinant M Protein of Porcine Reproductive and Respiratory Syndrome Virus

To set up a sensitive,specific,rapid and high throughput serological antibody detection method for porcine reproductive and respiratory syndrome virus(PRRSV),M protein were expressed using prokaryotic expression technology,an indirect ELISA antibody detection method for PRRSV were set up using the purified recombinant M protein as coating antigen. Based on published PRRSV M genome sequence,one pairs of specific primers were designed,amplified 435 bp M gene fragment by RT-PCR,the fragment was cloned into pET32a(+) expression vector,and induced by IPTG,the recombinant M protein was expressed in inclusion body forms,after recombinant protein purification,Western blotting showed the protein had good antigenicity and specificity.After optimization of reaction conditions,using the purified recombinant M protein as antigen established an indirect ELISA method for PRRSV,the established M-ELISA detected six kinds of swine disease (CSFV,JEV,TEDV,PRV,PPV,PCV2) positive sera were negative,the coefficient variation of the method intra-assay and inter-assay repetitive tests were less than 5% and 10%,compared with IDEXX ELISA kit, the coincidence rate of the method was 95.3%. The established M-ELISA detection method provided a simple,rapid,high throughput serological antibody detection method for the PRRS vaccinated pigs antibody level monitoring,and the wild PRRSV infection diagnosis and epidemiological investigation.