Recombination Human Papillomavirus HPV11E7Protein Expression And Its Polyclonal Antibody Preparation And Identification

ObjectiveCondylomata acuminate(CA) is one of the most common sexually transmitted diseases, there were reports that the risk of CA infection in sexually active individuals was as high as80%[1]. The disease often existed in the form of subclinical infection and had the characteristics of strong infectiousness. refractory and high recurrence rates[2]. caused great health and economic burden to patients. Human papillomavirus(HPV) type11was one of the low-risk subtypes of HPV. As HPV early gene E7protein played a very important role in persistent HPV infection and carcinogenesis, it was currently one of the major targets for HPV therapeutic vaccines. But there was no commercial HPV11E7proteins or antibodies at present, and the high epithelium and species specificity of HPV made it difficult to culture virus in vitro or establish infection model. So through molecular biological method to obtain a large number of HPV11E7protein and its polyclonal antibody might provide experimental basis for futher functional studies of HPV11E7protein as the target of immunointervention. The aim of this study was to construct prokaryotic expression vecter pGEX-4T2-HPV11E7. The recombinant vectors was transformed into E.coli JM109and a large amount of GST-HPV11E7fusion protein was prepared. Purified the protein and immunized New-Zealand rabbits, antiserum against HPV11E7was obtained. Identification the titer and specificity of purified Anti-HPV11E7polyclonal antibody IgG.Methods1.Construction and identification of HPV11E7prokaryotic and eukaryon expression vecterPrimer was designed to construct a prokaryotic expression vecter pGEX-4T2-HPV11E7. With HPV11purified plasmid DNA as a template, the products were amplified by PCR and connected to pMD(?)18-T Vector, then through EcoR I and Not I enzyme recovery the DNA was connected to pGEX-4T2Vector. After EcoR I and Not I enzyme identification and sequencing, the prokaryotic recombinant vecter was transformed into E.coli JM109.Primer was designed to construct a eukaryon expression vecter pEGFP-C1-HPV11E7. With HPV11purified plasmid DNA as a template, the products were amplified by PCR and connected to pMD(?)18-T Vector, then through EcoR I and BamH I enzyme recovery the DNA was connected to pEGFP-C1Vector. After EcoR I and BamH I enzyme identification and sequencing, the prokaryotic recombinant vecter was prepared to transfect HEK-293cells.2.Acquisition and identification of GST-HPV11E7fusion proteinPlasmid pGEX-4T2-HPV11E7was transformed into E.coli JM109and the transformants were cultured in LB medium with0.2mM IPTG in26℃for6hours to expess soluble fusion prtotein. The lysed transformants were analyzed by15%SDS-PAGE. The expressed fusion protein was purified via Glutathione-Sepharose4B column and thrombin cleavage in order to obtain HPV11E7protein.3. Preparation and detection of polyclonal antibody IgGPurified HPV11E7protein was used to imummize the New Zealand rabbits. After one primary immunization and three booster immunizations, the sera of rabbits were collected to detect the titer. When titer higher than1:8, serum was gained after ultimate immunization. According to the rProtein G Agarose instruction, purified HPV11E7polyclony IgG antibody was obtained.15%SDS-PAGE identified the titer of HPV11E7protein, with different dilution (1:200,1:400,1:800,1:16000,1:32000,1: 64000) HPV11E7polyclony IgG antibody as the first antibody, Goat anti rabbit-HRP as the two antibody, bovine serum albumin(BSA) as blank control.4. Identification the specificity of HPV11E7polyclonal antibodyTransient transfection of pEGFP-C1、pEGFP-C1-HPV11E7to HEK-293T cells, Western blot analysis with1:1000HPV11E7polyclony IgG antibody as the first antibody, goat anti rabbit-HRP as the two antibody. HEK-293T cells without transfection as blank control.Untransfected HEK-293T cells, pEGFP-C1and pEGFP-C1-HPV11E7transfected HEK-293T cells growed on glass coverslips. Dispose the cells with formalin fixation, Triton X-100incubation and goat serum blocking,1:500HPV11E7polyclony IgG antibody as the first antibody, Alexa Flour(?)555marked goat anti rabbit-HRP as the two antibody, immunofluorescence analysis the cells.Results1.Construction and identification of HPV11E7prokaryotic and eukaryon expression vecter1%agarose electrophoresis identified HPV11E7gene amplified by PCR, the result showed a clear band at310bp. The amplified fragments were cloned into pMD-18T vector and subclonedinto pGEX-4T2vector (eukaryotic was pEGFP-Cl). Enzyme identified the gene with EcoR I and Not I (eukaryotic was BamH I),1%agarose electrophoresis analysis showed a clear band at310bp. Sequencing pGEX-4T2-HPV11E7and pEGFP-C1-HPV11E7, the gene proved completely correct by BLAST comparison.2.Acquisition and identification of GST-HPV11E7fusion proteinPlasmid pGEX-4T2-HPV11E7was transformed into E.coli JM109cells, the lysed transformants were analyzed by15%SDS-PAGE. One protein band from supermant at molecular weight of37KD was detected, which was the same as the molecular weight of GST-HPV11E7protein from references., After purification via thrombin cleavage.15%SDS-PAGE analysis demonstrated a protein band at molecular weight of11KD, which was the same as the molecular weight of HPV11E7protein from references. 3. Preparation and detection of polyclonal antibody IgGOne sediment line appeared between the bores of HPV11E7protein and sera from immunized rabbit by double immunodiffusion assay, indicating that the potence of sera against HPV11E7protein was the value of1:32. The further purified HPV11E7polyclonal IgG antibody could specifically identify the HPV11E7protein by western blot assay, which demonstrated a specific band in the molecular weight about11KD. The titer of this antibody was more than1:64000.4. Identification the specificity of HPV11E7polyclonal antibodypEGFP-C1-HPV11E7transfected HEK-293T cells were used for western blot to detect the titer and specificity of HPV11E7polyclonal IgG antibody. The result showed a specific band in the molecular weight of about39KD which is consistent with GFP-HPV11E7fusion protein. pEGFP-C1transfected and untransfected HEK-293T cells did not show corresponding bands.Immunofluorescence on untransfected HEK-293T cells showed no green or red fluorescence. Green fluorescent protein EGFP could be expressed in pEGFP-C1and pEGFP-Cl--HPV11E7transfected HEK-293T cells. Specificity red fluorescence was expressed in plasmid pEGFP-C1-HPV11E7transfected HEK-293T cells. The result showed HPV11E7antibody could specifically recognize the HPV E7protein expressed in HEK-293T cells.Conclusions1. Successful expression and purification large amounts of soluble HPV11E7protein.2. Antiserum against HPV11E7was obtained in immunized rabbits and purified rabbit HPV11E7polyclonal IgG antibody was obtained.3. Purified anti-HPV11E7polyclonal antibody IgG showed high titer and specificity as confirmed by Western-Blot and immunofluorescence.4. HPV11E7protein provided experimental basis for futher functional studies of HPV11E7protein as the target of immunointervention. And the successful preparation of HPV11E7polyclonal IgG antibody might provide new test for clinical and scientific research.