猪圆环病毒2型Cap基因的原核表达及ELISA检测方法的建立

Expression of Cap gene of porcine circovirus type 2 in Escherichiia and development of a detection method of ELISA

目的:方法:为建立PCV-2 ELISA检测方法。利用PCR技术从PCV-2基因组中克隆Cap抗原表位基因,并将其插入到载体pET-22b(+)中,构建成原核表达质粒pET-22b(+)-Cap,使其在E.coliBL21(DE3)中以IPTG诱导表达并进行纯化,以表达的蛋白进行包被,建立ELISA检测方法。结果:表达产物分子质量约30 kD,经Western blot表明具有良好的免疫活性,对643份猪血清进行检测,阳性率为73.1%,阴性率为26.9%。结论:该检测方法能在临床中进行应用。

Objective：In this experiment, ELISA measurement was developed to detect antibody agaist PCV-2. Methods： A Cap sequence encoding for antigen epitope was obtained by PCR from the genome of porcine circovirus type 2 and then cloned into prokaryotic expression plasmids pET-22b（ ＋ ）.The plasmid of pET-22b（ ＋ ）-cap was successfully constructed and transformed into E. coli BI21（DE3）. An ELISA for detection of PCV-2 was established by high expression of Cap protein after induced by IPTG. Results： The results of Western blot showed that the expressed protein was 30 kD and possessed immunological activity. Positive rate was 73.1% and negative rate was 26.9% respectively for 643 serum samples which were examined with the measurement by ELISA. Conclusion： The ELISA method is apphcable in clinic.