摘要
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435bp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。
To set up a sensitive,specific, rapid and high throughput serological antibody detection method for porcine repro- ductive and respiratory syndrome virus（PRRSV）, M protein were expressed using prokaryotic expression technology, an indi- rect 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 anti- genicity and specificity. After optimization of reaction conditions, using the purified recombinant M protein as antigen estab- lished 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 repeti- tive 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.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第7期51-55,共5页
China Animal Husbandry & Veterinary Medicine
基金
滨州职业学院科学研究基金项目(10xykt06)
关键词
猪繁殖与呼吸综合征病毒
重组M蛋白
间接ELISA
抗体检测
porcine reproductive and respiratory syndrome virus
recombinant M protein
indirect ELISA
antibody detection
