猪细小病毒VP2蛋白的主要抗原表位基因的原核表达及检测应用

Prokaryotic Expression and Detective Application of the Main Antigenic Region of VP_2 of Protein Porcine Parvovirus

将构建好的重组质粒PET-VP2Ⅰ转化宿主菌BL21,利用IPTG(1mmol/L)诱导实现了PET-VP2Ⅰ蛋白主要抗原域VP2Ⅰ融合蛋白的高效表达。通过Western-blot检测证明表达的重组蛋白具有良好的免疫学活性。表达产物经HisBind层析柱纯化后作为诊断抗原初步建立了检测PPV抗体的间接ELISA方法。结果表明:抗原的最佳包被浓度为3.5μg/mL,血清的最佳稀释度为1∶40,待检血清阳性标准初步定为OD490>0.51,且待检血清的OD490值与阴性血清的OD490值的比值大于2.1。

The recombinant plasmid pET-VP2 Ⅰ was transformed into BL21 competent cells and expressed in high level after induced with 1.0mmol/1 IPTG. The expressed product was purified with His·Bind chromatograghy after being proved by Westen-blot,and was used as an antigen to establish indirect ELISA for detecting antibodies against PPV. The optional working circumstances for the ELISA（antigen concentration 3. 5/μg/mL; serum dilution 1：40 ）were tried out with chess titration. The positive critertion of test serum of this ELISA is OD490nm〉0.51 ,the OD490 ratio of the tested serum to the negative serum is higher than 2.1.