Expression Of ORF50 Truncated Genes, VIL-6 And GL Full Length Genes Of Human Herpesvirus 8 And Identification Of Polyclonal Antibodies Against Peptides Of ORF50 And VIL-6 Proteins

Human herpesvirus 8 (HHV-8) , also known as Kaposi's sarcoma-associated herpesvirus (KSHV) , was a human gamma-2 herpesvirus, which was associated with the pathogenesis of Kaposi's sarcoma (KS), primary effusion lymphomas (PEL), and multicentric Castleman's disease (MCD). HHV-8 ORF50 (open reading frame 50) gene was considered to be the molecular switch gene controling the latent and lytic replication. HHV-8 vIL-6 (viral interleukin-6) gene, a homologue of human IL-6 gene, was involved in cell proliferation and HHV-8 gL (glycoprotein L) gene participated in virus absorption and entering. In this study, three ORF50 truncated genes and the full length vIL-6 and gL gene of HHV-8 were correctly cloned and expressed in E.coli. The vIL-6/GST fusion protein was successfully purified. Furthermore, polyclonal antibodies against peptides of ORF50 and vIL-6 were prepared and identified.1. Cloning and expression of ORF50 truncated genes, the full length vIL-6 and gL genes of HHV-8 in E.coli.Three pairs of PCR primers were designed according to 3'ends of ORF50 gene encoding C-terminal amino acids (aa 525-691, aa 513-660, aa 541-691) and a pair of PCR primers was also designed according to the full length vIL-6 and gL gene, respectively. The target genes were amplified by PCR taking plasmids pcDNA3.1+ORF50 or pcDNA3.1+vIL-6 or pcDNA3.1+gL as templates. Amplified PCR fragments were subcloned into the prokaryotic expression vector pGEX-6p-1 to construct five recombinant expression plasmids. After identification with endonuclease digestion and nucleotide sequences analysis, the recombinant prokaryotic expression plasmids were transformed into host BL21 (DE3) cells. The GST fusion protein expression was successfully induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and detected by SDS-PAGE and Western blot.2. Denaturating and renaturating of the gL fusion protein inclusion body and purifing both gL and vIL-6 fusion proteinsDenaturating the gL fusion protein inclusion body by high density urea and renaturating by dialyzing steply at low temprature, the soluble gL fusion protein was obtained. The vIL-6 and gL fusion protein were purified by affinity laminar analysis through chromatography Glutathione Sepharose 4B. The vIL-6 fusion protein was successfully purified and detected by SDS-PAGE and Western blot.3. After estimating the protein secondary structure and B cell epitopes, polyclonal antibodies against peptides ORF50 and vIL-6 proteins were prepared and identified.After estimating the ORF50 C-terminal amino acids and the full length vIL-6 protein secondary structure and B cell epitopes by DNAStar Protean software, peptides located in 527～539 and 667～691aa of ORF50 protein and 89～101 and 137～148aa of vIL-6 protein were synthesized and coupled with keyhole limpet hemacyanin (KLH). Then the peptides were used to immunize rabbit to obtain polyclonal antibodies. Western blot analysis was performed to examine the characteritics of the polyclonal antibodies.