山羊关节炎-脑炎病毒抗体间接ELISA检测方法的建立

Development of an indirect ELISA for the detection of antibodies against caprine arthritis encephalitis virus using recombinant CA protein

为建立检测山羊关节炎-脑炎病毒(CAEV)血清抗体的间接ELISA方法,采用PCR方法扩增山羊关节炎-脑炎病毒CA蛋白编码基因序列,然后克隆至原核表达载体pET-28a(+),诱导含有重组载体pET-28a(+)-CA的大肠杆菌BL21(DE3)表达重组蛋白rHis-CA。采用Ni柱亲和层析方法纯化重组蛋白rHisCA,并以纯化的rHis-CA为包被抗原建立检测山羊关节炎-脑炎血清抗体的间接ELISA方法。结果表明重组蛋白rHis-CA以包涵体形式表达,具有良好的反应原性。所建立的rHis-CA间接ELISA方法的最适抗原包被质量浓度和血清稀释度分别为0.4 μg/mL和1∶100,血清阴阳性的D_(450)临界值为0.45。该方法仅与山羊关节炎-脑炎病毒阳性血清发生特异性反应,不与山羊痘病毒、蓝舌病病毒和口蹄疫病毒阳性血清发生交叉反应。对来自陕西地区的48份山羊血清进行CAEV抗体检测,rHis-CA ELISA和琼脂凝胶免疫扩散试验的阳性检出率分别为35.41%(17/48)和31.25%(15/48)。对来自天津地区2个山羊场的240份山羊血清样本进行CAEV抗体检测,rHis-CA ELISA方法和琼脂凝胶免疫扩散试验的检测结果均为阴性。

In order to develop an indirect ELISA for the detection of antibodies against caprine arthritis encephalitis virus（CAEV） using recombinant CA protein,the CA gene was amplified by polymerase chain reaction,and then the amplified DNA fragment was cloned into an expression vector, pET-28a（＋）.Whe recombinant bacterial（BL21） cells harboring plasmid pET-28a（＋）-CA were induced to express recombinant protein rHis-AC. The recombinant protein tHis-At was purified by affinity chromatography using Ni-NTA column. The rHis-CAELISA for detection of antibodies against CAEV was determined by optimazing conditions using purified tHis-At as coating antigen. The results showed that the rHis-CA was expressed as inclusion bodies and had good reactionogenicity. The optimal concentration of coating antigen and dilution of serum were 0. 4 μg/mL and 1：100,respectively. The cut-off value of ne- gative and positive sera was 0.45. The rHis-AC ELISA method exhibited good specificity,and showed no cross-reactions with positive sera of sheeppox virus,bluetongue virus and foot-and-mouth disease virus. Of the 48 goat serum samples from Shaanxi,35.41%（17/48） were positive 31.25%were positive in agar gel immunodiffusion（AGID）. All 240 goat serum samples from Tianjin were negative by rHis-CA ELISAand AGID.