目的 制备人疱疹病毒7型(HHV-7)pp85蛋白及其抗体。方法 采用聚合酶链反应(PCR)方法自HHV-7型YY5分离株中扩增出pp85基因,经测序后构建原核表达质粒PGEX-6p-1+pp85b。重组质粒转化大肠埃希菌Rosetta,诱导蛋白表达。应用酶切鉴定、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及蛋白质印迹法等方法确保基因片段的正确性及表达蛋白的特异性。表达的蛋白经亲和层析纯化后,免疫动物制备多克隆抗体,并通过免疫荧光法鉴定抗体的特异性。结果 成功地获得了高纯度的pp85融合蛋白,纯度可达90%以上;免疫动物后制备的多克隆抗体效价可达1∶102400,该抗体能特异识别HHV-7抗原,而不与HHV-6反应。结论 获得高纯度的HHV-7pp85融合蛋白及其高效价的抗体,将进一步应用于临床检验。

Objective The goal of the current study was to express the pp85 gene of human herpesvirus 7 (HHV-7) and to prepare anti-pp85 polyclonal antibodies. Methods The pp85 gene of HHV-7 was amplified using PCR and this amplified gene was cloned into a prokaryotic expression vector and named as PGEX-6P-1+pp85b. The recombinant plasmid was then transformed into Escherichia coli Rosetta. The accuracy of the inserted gene and the specificity of the proteins were verified by restriction enzyme digestion,SDS-PAGE,and Western blot. The expressed pp85 fusion protein was then purified using affinity chromatography and was used to immunize rabbits in order to obtain the anti-pp85 antibodies. The specificity of these antibodies was verified using immunofluorescence. Results The purity of the recombinant pp85 was above 90% and the titer of anti-pp85 antibodies was 1∶102 400. As an indication of the specificity of the antibodies produced,this polyclonal antibody was shown to react with HHV-7 infected cells but failed to react with human herpesvirus 6 (HHV-6) infected cells as shown by IFA.Conclusion In the current study we were able to obtain a highly purified form of the HHV-7 pp85 fusion protein and the corresponding high-titer polyclonal antibodies were produced in rabbit. if(document.getElementById('ChDivSummary').innerHTML!=""){CutSpan('ChDivSummary',500);DisplaySpanDiv('ChDivSummary');ClearSummaryOnLoad('SummaryLinkChID','SummaryLinkEnID');}else{CutSpan('EnDivSummary',1000);DisplaySpanDiv('EnDivSummary');ClearSummaryOnLoad('SummaryLinkEnID','SummaryLinkChID');}