| Objectives1. To detect and type HPV DNA in peripheral blood mononuclear cells ( PBMCs) and in local lesions of patients with condyloma acuminata ( CA). To find out the relationship between the HPV DNA detected in PBMCs and those in local lesions.2. To obtain HPV6 major capsid protein encoded late gene one (L1) with baculomavirus expression system.3. To detect the HPV6 L1 IgG antibody in the sera of patients with CA and normal blood donors, using the obtained HPV6 L1 protein as antigen.Materials and Methods1. collecting samples and extracting DNAWe collected the blood and biopsy samples of 30 CA, confirmed by clinical manifestation, acetowhitening test and pathology. Blood samples collected from 20 age - matched normal dornors served as controls. The blood samples were collected in heparin and PBMCs were fractioned by density gradient centrifuga-tion in lymphocyte separation medium. Total DNA from PBMCs, plasma and local lesions were extracted with phenol and chloroform.2. PCR amplification and HPV genotypingIn order to identify HPV DNA in the local wart, PBMCs and plasma of CA patients, the PCR was employed using the HPV L1 consensus primers MY09 and MY11.The HPV genotypes of the MY09/MY11 PCR products from verrucous lesions and PBMCs were determined by restriction fragment length polymorphisms( RFLP) technique, with two restriction enzymes, Rsa I and Pst I, respectively. The digested PCR fragments were separated by electrophoresis.3. Sequence analysis of the PCR productsTo confirm the HPV types identified by the RFLP, the purified PCR products from three patients were ligated into the vector pGEM - T and verified by sequence analyses. The obtained sequence was compared with the published HPV sequences in Genbank.4. Cloning and sequencing of HPV6 LI geneWe designed the HPV6 specific primer to amplify the LI gene, and introduced restriction enzymes sites for BamHI and EcoRI into the primer. DNA extracted from CA verrucous lesion, which was verified as HPV type 6 by RFLP, was employed as template to earn' out PCR amplification. The products were ligated to the vector pGEM - T and sequenced.5. Construction of pFASTBacHTb - HPV6L1 donor plasmidThe cloned plasmid on vector pGEM - T was digested with BamHI and Eco-Rl. The HPV6 LI DNA fragments were ligated to the pFASTBacHTb vector that had already been enzymed with BamHI and EcoRI. Then the recombinant pFASTBacHTb - HPV6L1 donor plasmid was constructed for further use.6. Transposition and isolation of recombinant Bacmid DNAThe pFASTBacHTb - HPV6L1 donor plasmid was transformed into E. coli. DH10BAC for transposition into the Bacmid, then added SOC medium to the mixture. The mixture was placed in a shaking incubator at 37℃ for 4h. The mixture was then diluted to 10-1, 10-2, 10-3. Each dilution was placed on the Luria Agar plates and incubated 36 ~48h at 37@. White colonies were selected and placed in a liquid culture. The liquid culture was placed in shaking incubator for 24h at 37@, and then recombinant Bacmid DNA was isolated.7. Transfection of Sf9 cells and harvest of recombinant baculovirusThe transfection was carried out in SFM culture without antibiotics, and the complex of liposome and Bacmid DNA was transfected into the Sf9 cells. The recombinant baculovirus from cell culture medium were then harvested at 72h post - transfection.8. Identification of HPV6 LI DNA and recombinant proteinThe DNA and proteins of the Sf9 cells infected recombinant baculovirus were extracted and identified by PCR, SDS -PAGE and Western - blot.9. Purification of recombinant protein.The Sf9 cells infected recombinant baculovirus were split by guanidine hydrochloride. The acquired supernatant was added to pre - equilibrated column. The column then was washed with different imidazole concentration buffer liquid. The eluent was collected and investigated by SDS - PAGE and Western - blot.10. Detection of HPV 6 L1 IgG antibody in serum by ELISAUsing the purified HPV6 L1 protein as the antige... |
