Construction And Application Of The Expressive Clone Of Human Herpes Virus 6 101K Tegument Protein

Objective To construct prokaryotic expression clone of human herpes virus 6 (HHV-6) 101K protein gene, for the preparation of efficient recombinant antigen for serological diagnosis of HHV-6 active infection.Methods The HHV-6 virus strain was isolated by cell culture from clinical saliva specimens. The virus strain that resulted in cytopathic effect was confirmed by PCR with a pair of HHV-6 specific primer and restricted endonuclease Hind III digestion. A fragment coding for HHV-6 major antigen epitopes (666-843 aa) was amplified by PCR technique. The PCR products were cloned into pCR2. 1 TA cloning vector for sequencing and subsequently compared with the sequence of HHV-6B Z29 strain in Genebank. The target gene was inserted into the prokaryotic expression plasmid pThioHis A. After the transformation of E. coli BL 21, the recombinant plasmid was induced with IPTG to express the 101K fusion proteins and the products were purified by nickel-chelating Sepharose resin affinity chromatography. The antigenicity and the specificity of the recombinant proteins was identified by Western blot. ELISA coating with the recombinant proteins was used to screen 197 sera, which simultaneously detected by indirect immunofluorescene assay (IFA). The cross-reactivity with human cytomegalovirus (HCMV) was also detected.Results The sequence of the target gene was identical with that of HHV-6B Z29 strain in Genebank. HHV-6 101K fusion proteins were efficiently expressed in E. coli BL21. In Western-blot assay, the fusion proteins reacted with 10 of 12 (83. 3%) HHV-6 IgM-positive serum samples and none of the 8 negative serum samples. ELISA coating with the recombinant proteins had a good agreement (95.4%) with commercial IFA kit, also had no cross-reactivity with HCMV positive serum.Conclusion The HHV-6 101k fusion proteins remain good antigenicity and specificity. It could be used in the serological diagnosis for acute and active HHV-6 infection after further perfected and provided useful information for development of HHV-6 recombinant antigens.