摘要
采用PCR方法扩增猪圆环病毒2型(PCV-2)Rep蛋白基因,克隆入原核表达载体pET-32a(+),转化大肠杆菌BL21构建重组表达菌,经SDS-PAGE检测目的蛋白得到成功表达,采用His标签单抗和PCV-2阳性猪血清进行Western blot鉴定表明重组蛋白具有良好的抗原性。利用纯化的重组蛋白rRep作为包被抗原,通过反应条件优化,建立了间接ELISA抗体检测方法。抗原最适包被质量浓度为1mg/L(0.1μg/孔),待检血清最适稀释度为1∶100,作用时间为1h,酶标抗体最适稀释度为1∶3 000。用该方法检测猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)阳性血清,结果均为阴性;批内和批间重复试验变异系数分别为<5%和<10%,表明本方法具有较好的特异性和重复性。应用rRep-ELISA对113份血清样品进行检测,与rCap-ELISA结果比较,rRep-ELISA检测阳性率为50.44%(57/113),略低于后者的54.87%(62/113),2种方法检测符合率为88.5%(100/113),具有较好的一致性。这为PCV-2感染的流行病学调查和新型ELISA诊断试剂盒的研制奠定基础。
The gene of and cloned into pET porcine circovirus type 2 32a （^-） vector. After （PCV-2） Rep protein gene was amplified by PCR, identification of positive recombinant plasmid and transformation, the recombinant bacteria BL21-Rep were obtained. SDS-PAGE and Western blot a-nalysis showed that recombinant protein （rRep） was expressed successfully and possessed good antigenicity. An indirect ELISA was developed for detection of PCV-2 antibodies with purified rRep protein. The optimal concentration of coating antigen was 1 mg/L （0.1℃g/well）. The serum sample was diluted by 1 ： 100 and incubated for 1 h at 37℃. HRP conjugated rabbit anti-pig IgG was diluted by 1 ： 3 000. Coating antigen had no cross reaction with the antibodies against CSFV, PRRSV and PRV. The intra-batch and inter-batches variation coefficients were lower than 5 % and 10%,respectively. 113 serum samples were tested with the rRep-ELISA, together with rCap-ELISA established in our lab. The positive rate of rRep-ELISA was 50.44%, lower than that of rCap-ELISA （54.87%）. The total coincidence rate was 88.5%. It might be useful for epidemiolog-ical investigation of PCV-2 infection and development of new diagnositic method.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第8期1154-1158,共5页
Chinese Journal of Veterinary Science
基金
江苏省农业科技自主创新基金资助项目(SCX(11)2143)
