人巨细胞病毒pp150-pp65蛋白的表达与应用

The fusion expression and application of pp150-pp65 of HCMV

目的构建并表达人巨细胞病毒(HCMV)pp150-pp65蛋白,将纯化的重组蛋白制备成胶体金试剂盒对其抗原性进行评价。方法采用PCR方法扩增HCMV pp150-pp65基因片段,连接到原核表达载体pET28a进行表达并纯化,采用SDS-PAGE和Western blot进行鉴定,并与北京英诺特生物技术有限公司合作研制出巨细胞IgG/IgM抗体联合检测试剂盒(胶体金法),与进口试剂盒(酶免法)进行临床标本比对试验。结果成功构建了HCMV的高效表达载体pET28a-pp150-pp65,并得到分子量在45kd的高表达蛋白,具有良好的抗原性。将该蛋白制备成胶体金试剂盒,经600份血清标本的检测,与进口CMV-IgG试剂盒进行比对,本试剂盒的阳性符合率和阴性符合率分别为92.7%和83.1%,总的符合率为90.2%;与进口CMV-IgM试剂盒进行比对,本试剂盒的阳性符合率和阴性符合率分别为88.1%和89.2%,总的符合率为88.5%,稳定性良好。结论高效表达纯化的pp150-pp65重组蛋白抗原性强,利用其建立的胶体金试剂盒与国产已上市试剂盒和进口试剂盒比对,不仅具有较高的灵敏度和特异性,而且经初步临床应用,表明该试剂盒能检测不同临床感染阶段的患者,具有较好的市场前景。

Objective To construct and express the recombinant plasmid and use the purified protein for the colloidal gold kit for its antigenicity evaluation.Methods Using PCR technology,the pp150 and pp65 gene fragments were cloned and expressed,The recombination protein was induced,purified and identified.At last to construct the combined detection Kit for IgG/IgM Antibody to HCMV（Colloidal Gold） to detect the samples in contrast with the imported reagent kit.Results The gene fragment was correctly amplified and successfully construct the recombinant vector.The purified protein with the molecular weight of 45 kd had good antigenicity.The protein was assembled into a colloidal gold kit to detect 600 serum samples,in contrast with the imported reagent kit HCMV-IgG,the kit of the sensitivity was 92.7%;specificity was 83.1%,coarse consistency was 90.2%;in contrast with the imported reagent kit HCMV-IgM,the kit of the sensitivity was 88.1%,specificity was 89.2%,coarse consistency was 88.5%;in contrast with the local reagent kit,coarse consistency to IgG and IgM was 93.0% and 91.2%,the stability is good.Conclusion HCMV pp150-pp65 protein highly expressed had strong specificity.Contrast to the local and the imported reagent kit,the colloidal gold kit had high sensitivity and specificity.It could distinguish the patients of different clinical infective stage,so it has a good market prospect.