The Preparation Of Anti-HPV6b E7protein Antibody And Culture Of Keratinocytes From Condyloma Acuminatum

Background:Low-risk or high-risk human papillomavirus (HPV) subtypes infection is very common. HPV6b, one of the low-risk subtypes of HPV, usually induces infection in keratinocytes of skin or epithelium of anogenital mucous membranes, predominantly generating benign squamous epithelial lesions commonly known as benign warts. Local immune suppression and/or abnormality in HPV protein recognition, which lead to HPV immune evasion and persistent infection are the main reasons for therapeutic resistance to HPV-associated diseases. Therefore, studying HPV immunopathogensis and the possible intervention strategies will be helpful to seek more effective therapeutic regimen as well as preventive methods for HPV infection. As HPV early gene E7protein plays a very important role in persistent HPV infection and carcinogenesis, it is currently one of the major targets for HPV therapeutic vaccines.The prepatation of the anti-HPV6b E7protein antibody may provide the experimental basis for futher functional studies of HPV6b E7protein as the target of immunointervention. Moreover, successful culture of HPV gene-expressing human keratinocytes from condyloma acuminatum will be extremely useful in futher investigation of the immunopathogenesis of HPV-associated disease.Objective:The aim of this study was to prokaryotically express and prepare polyclonal antibody of HPV6b E7protein on the basis of our previous investigation. In anddition, we supposed to culture and identify the human keratinocytes from foreskin and genital warts.Methods:Plasmid pGEX-4T-2/HPV6b E7was transformed into E.coli DH5a and induced by IPTG to expess soluble fusion prtotein. Then, the transformants were lysed by sonicator. And the products were analyzed by SDS-PAGE. The expressed fusion protein was purified via Glutathione-Sepharose4B column and thrombin cleavage in order to obtain HPV6b E7protein. Subesquently, HPV6b E7protein was used to imummize the New Zealand rabbits. The sera of rabbits were collected and purified to HPV6b E7polyclony IgG antibody. Human keratinocytes isolated from foreskin and genital warts were cultured in modified method. The DNA of human keratinocytes were extracted to identify the HPV subtypes via PCR assay.Results:1. Induction of GST-HPV6b E7fusion protein, purification and identification of HPV6b E7protein.Plasmid pGEX-4T-2/HPV6b E7was transformed into E.coli DH5acells and the transformants were cultured in LB medium with0.2mM IPTG in28℃for4hours. The lysed transformants were analyzed by12%SDS-PAGE. One protein band from supermant at molecular weight of37KD was detected. After purification via Glutathione-Sepharose4B column and thrombin cleavage, SDS-PAGE analysis demonstrated a protein band at molecular weight of14KD. which was the same as the molecular weight of HPV E7protein from references.2. Preparation and detection of polyclonal antibody IgGOne sediment line appeared between the bores of HPV6b E7protein and sera from immunized rabbit by double immunodiffusion assay, indicating that the potence of sera against HPV6b E7protein was the value of1:8. The further purified HPV6b E7polyclonal IgG antibody could specifically identify the HPV6b E7protein by western blot assay, which demonstrated a specific band in the molecular weight about14KD. The titer of this antibody was more than1:5000.3. Identification the specificity of HPV6b E7Polyclonal antibodyHEK293T cells and B16cells were successfully transfected with recombinant plasmid pcDNA3.1(+)-GFP/HPV6b E7. Protein extracted from pcDNA3.1(+)-GFP/HPV6b E7transfected HEK293T cells were used for western blot to detect the titer and specificity of HPV6b E7polyclonal IgG antibody. The result showed a specific band in the molecular weight of about41KD which is consistent with GFP-HPV6b E7fusion protein. Immunofluorescence on pcDNA3.1(+)-GFP/HPV6b E7transfected HEK293T cells and B16cells also showed that HPV6b E7antibody could specifically recognize the HPV E7protein expressed in pcDNA3.1(+)-GFP/HPV6b E7transfected HEK293T cells and B16cells.4. Culture and identification of human keratinocytes isolated from condyloma acuminatumHuman keratinocytes derived from foreskin and genital warts were successfully cultured in a modified culture system. The PCR tests showed HPV DNA were expressed in human keratinocytes from genital warts. Conclusions:1. We have successfully expressed and purified amount of HPV6b E7protein.2. The purified rabbit HPV6b E7polyclonal IgG antibody were obtained.3. HPV6b E7polyclonal IgG antibody can specifically recognize the HPV6b E7protein expressed in tranfected HEK293T Cells and B16cells.4. Human keratinocytes isolated from foreskin and genital warts were successfully cultured. HPV DNA were identified in warts-derived keratinocytes by PCR assay.