人巨细胞病毒磷蛋白65及糖蛋白B在大肠杆菌中的高效表达、免疫原性研究及初步应用

实验成功的构建了含有pp65与gB主要抗原决定簇基因的表达质粒pET-pp65、pET-gB;表达产物通过SDS-聚丙烯酰胺凝胶电泳、免疫印迹、间接酶联免疫等一系列鉴定试验进行了分析。表达蛋白经初步纯化后免疫BALB/c小鼠,经0、2、4周免疫三次,免疫后2、4、6周眼眶采血收集抗血清,进行中和试验和ELISA检测。动物试验表明两种表达蛋白具有免疫原性,并可在小鼠体内诱导产生特异性抗体,其中用重组gB蛋白免疫小鼠后得到的抗血清在体外试验中能抑制天然病毒感染细胞。同时将这两种表达蛋白作为诊断用抗原,对临床94份血清进行检测,证实pp65具有较好的特异性,为今后研制HCMV诊断试剂提供了科学依据。

Expression and characterization of antigenic domains of human cytomegalovirus -pp65 and gB in E.coli

In this study,we successfully constructed expressing plasmids pET-pp65 and pET-gB,which contain genes of HCMV-pp65 and the gB coding for the main antigenic domains.These expressed proteins were analyzed by SDS-PAGE,Western-blot and indirect ELISA respectively.BALB/c mice were immunized by the expressed proteins in 0,2 and 4 weeks respectively,and collected antiserum in 2,4 and 6 weeks after each immunization.The determination of antibody titers were carried out by virus neutralization test and ELISA.These recombinant proteins show good immunogenicity,especially anti-gB serum is able to neutralize nature human cytomegalovirus in vitro.The recombinant protein pET-pp65 has a better specificity than that of pET-gB after 94 clinical serum samples were tested by using of two expressed proteins as diagnostic antigens.