弓形虫表面抗原P22的重组体构建及临床应用研究 [中文引用][英文引用]

目的 克隆并构建pGEX-4T-1-P22重组表达载体，获取纯化的P22蛋白，为进一步研究P22的生物学特性和免疫保护作用奠定实验基础。方法 应用PCR技术扩增P22基因片段，连接到载体中，诱导并纯化P22蛋白，进行SDS-PAGE和Western blot鉴定，并组装成弓形虫IgM 抗体酶联免疫（ELISA）试剂盒与进口试剂盒对比。结果 成功构建了重组质粒pGEX-4T-1-P22，并得到分子量在43KD的大量表达蛋白，具有良好的抗原性，将该蛋白组装成ELISA试剂盒检测IgM抗体，与意大利SORIN试剂盒对比检测450份临床血清，阴阳性符合率分别为92.16%和88.06%，总的符合率为88.89%；结论 高效表达纯化的P22重组蛋白抗原性强，利用其建立的IgM ELISA试剂盒与进口试剂盒对比，具有较高的灵敏度和特异性，可用于检测弓形虫抗体。

Objective To clone the p22 Protein Fragment and construct the recombination expression vectors, get the purified P22 protein, so as to lay an experimental foundation for the further study on the biological characteristics and immune protective effect. Method Using PCR technology，the P22 gene fragment was were cloned and expressed; the purified protein and indentified by SDS-PAGE and Western blot. Results  The gene fragment was correctly amplified and successfully construct the recombinant vector. The purified protein with the molecular weight of 43KD had good antigenicity. The protein was assembled into a ELISA kit to detect 450 serum samples, in contrast with the imported reagent kit Tox-IgM, the kit of the sensitivity was 92.16%; specificity was 88.06%, coarse consistency was 88.89%.Conclusions  p22 protein highly expressed had strong specificity. Contrast to the imported reagent kit,the kit had high sensitivity and specificity. It could test the antibody to TOX.