人疱疹病毒8型感染的间接ELISA检测方法的建立

目的〓〖HTK〗建立人疱疹病毒8型（HHV-8）感染的间接ELISA检测方法，并初步探讨其应用价值。〖HTW〗方法〓〖HTK〗利用PCR技术获得HHV-8 ORF73C基因，插入到原核表达载体pGEX-6P-1中，在大肠埃希菌BL21(DE3)中诱导蛋白表达；以纯化的GST-ORF73C融合蛋白为包被抗原，利用方阵滴定法确定包被抗原和抗血清的最适工作浓度，并通过阻断试验、交叉试验、特异性试验、重复性试验及与Chou等建立的K8.1合成多肽-ELISA检测方法进行试验比较等评价其效果。〖HTW〗结果〓〖HTK〗SDS-PAGE及Western-blot检测结果显示，目的基因在大肠杆菌BL21(DE3)中得到高效表达，且表达产物能被KS阳性血清［HIV(+)/KS(+)血清］特异性识别；方阵滴定试验显示：包被抗原的最佳浓度为1.5μg/mL，血清抗体的最佳稀释度为1∶80；阻断试验、交叉试验、特异性试验、重复性试验等表明该方法特异性强、重复性好；对86份HIV（+）/KS（-）血清样品进行检测的结果显示，该方法与Chou等建立的K8.1合成多肽-ELISA检测方法的符合率为98.8％。〖HTW〗结论〓〖HTK〗GST-ORF73C-ELISA检测方法具有良好的特异性和重复性，可用于HHV-8感染的血清学检测及流行病学调查。

Establishment of an indirect-ELISA method for detection of HHV-8 infection

Objective〓〖WTBZ〗To establish an indirect-ELISA method for detection of HHV-8 infection and to evaluate its preliminary application significance. 〖WTHZ〗Methods〓〖WTBZ〗The HHV-8 ORF73C gene was amplified with PCR and then was inserted into the prokaryotic-expression vector pGEX-6P-1. The GST-ORF73C fusion protein was expressed and purified from E.coli BL21(DE3) cells. The optimal dilution of coating antigen and serum samples was determined by using the chess titration test. The effect of the ELISA was estimated by an interruption test, a cross-reaction test, a specificity test, and a repetition test, and was compared with that of the K8.1 peptid-ELISA established by Chou et al. 〖WTHZ〗Results〓〖WTBZ〗SDS PAGE and Western blot analysis showed that the target gene was efficiently expressed in E.coli, and the expressed products showed a fine immunol-reactivity. A chess titration test showed that the optimal concentration of coating antigen was 1.5μg/mL and the optimal dilution of serum samples was 1∶80. An interruption test, cross-reaction test, specificity test and repetition test showed that the ELISA assay had good reproducibility and specificity. 86 serum samples were detected by both GST-ORF73C-ELISA and K8.1 peptide-ELISA, and the coincidence rate of the two methods was 98.8％. 〖WTHZ〗Conclusions〓〖WTBZ〗The GST-ORF73C-ELISA method is specific and repeatable, and is a good method for serological detection and epidemiological analysis for HHV-8 infection.