Construction And Application Of The Expressive Clone Of Human Herpesvirus 8 Small Capsid Protein Encoded By ORF65

Objective To construct prokaryotic expression clone of human herpes virus 8 (HHV8) small capsid protein encoded by ORF65, and to prepare of efficient recombinant antigen for serological diagnosis of HHV8 active infection.Methods BCBL-1 cells (HHV8-positive but EBV-negative) were cultured and harvested. HHV8 ORF65 coding sequence of the major antigen epitopes was amplified by PCR and inserted into the prokaryotic expression vector pThioHis A , recombinant plasmid was transformed into E.coli BL21 and induced to express by IPTG. The expressed products were purified by affinity chromatography on Ni-NTA resin. The antigenicity and the specificity of the recombinant proteins was identified by Western blot. ELISA coating with the cells lysates and the recombinant proteins was used to screen 568 serum samples, which were simultaneously detected by IFA kit (Biotrin) and ELISA kit (Qiagen) . The specificity, sensitivity, Youde-n's index, predictive value positive and predictive value negative of the cells lysates and the recombinant proteins were detected The cross-reactivity with EBV was also detected.Result ELISA coating with the recombinant proteins had a good agreement with IFA kit (Biotrin) and ELISA kit (Qigen) , and had no cross-reactivity with EBV. The ELISA coating with the recombinant proteins showed high specificity and sensitivity in the detection of HHV8-specific IgG and IgM antibodies in clinical samples.The recombinant proteins had more specific and lower sensitive than the cells lysates.Conclusion The recombinant proteins can be used in the serological diagnosis for acute and active HHV8 infection. Recombinant prokaryotic expression vector was gained, and it is valuable for advanced study on HHV8 specific antibody detection.Postgraduate: Xing-dong Ju(Pathogenic Organism)Directed by: Bing Luo...