Prokaryotic expression and application of human herpes virus 8 small capsid protein encoded by open reading frame 65 / 中华检验医学杂志

ABSTRACT

Objective To construct prokaryotic vector and express human herpes virus 8 (HHV8) small capsid protein open reading frame 65 (ORF65) in Escherichia coli.Methods DNA was extracted from BCB-1 cells (HHV8-positive but EBV-negative).HHV80RF65 coding sequence was amplified by PCR and inserted into the prokaryotic expression vector pThioHisA.Recombinant plasmid was transformed into E.coli BL21 to express fusion protein induced by IPTG.The expressed products were purified by affinity chromatography on Ni-NTA resin.The antigenieity and the specificity of the recombinant proteins was identified by Western blot with HHVS-positive serum samples.ELISA coating with the recombinant proteins was used to screen 568 serum samples,which were simultaneously detected by IFA kit(Biotrin) and ELISA kit(Qiagen).Results Gene sequencing showed that the target gene was identical with that of HHV8 standard species,the 31 500 fusion protein was found in SDS-PAGE.ELISA coating with the recombinant proteins had a good agreement with IFA kit(Biotrin) and ELISA kit(Qiagen).The detection for the clinical samples showed the ELISA kit was feasible,applicable and consistant.Conclusion The recombinant proteins showed good antigenicity,and it is valuable for further study on HHV8 specific antibody detection.