关键词: 酶联免疫吸附测定, ORF73C基因, 人疱疹病毒8型

Abstract:

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.

Key words: HHV-8, ORF73C, Enzyme-linked immunosorbent assay