Cloning And Expression Of Human Papillomavirus Type 16 E6/E7 Gene In Lanzhou City

Background and objective:HPVs are small, non-enveloped viruses that contain circular double-stranded DNA.118 different genotypes of HPVs have been identified, of which about 40 infect the genital mucosa. These viruses can be classified into high-risk or low-risk groups according to the propensity for malignant progression of the associated lesions which they cause. High-risk HPVs, such as HPV16,18 and 31, are associated with more than 90% of cervical cancers, and of which HPV16 is the most. Two early oncogenes E6 and E7 are invariably expressed in cervical cancer and more than 54% of cervical intraepithelial neoplasia (CIN)â…¡-â…¢, so E6 and E7 proteins are closely related to the occurrence and development of cervical cancer. This study investigates on the cloning of HPV type 16 E6 and E7 gene, and expression of E6 and E7 proteins in E.coli. This is a basic study for the protein vaccine on animal, the molecular mechanism of cervical cancer induced by E6 and E7 oncogenes and immunologic therapy of cervical cancers and precancerous lesions in future.Methods:1. PCR technique was used to amplify the HPV16 E6 and E7 genes fragment from the DNA of cervical cancer tissue;2. HPV 16 E6 and E7 gene fragments were cloned into pMD 19-T Simple vector to construct cloning plasmids pMD 19-T-E6, pMD 19-T-E7;3. The cloning plasmids pMD 19-T-E6, pMD 19-T-E7 were digested with EcoR I and Hindâ…¢, then the HPV16 E6 and E7 genes were cloned into the expression vector pET28a(+) to construct expressing plasmids pET28a(+)-E6 and pET28a(+)-E7, using the T4 DNA ligase;4. The recombinant expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 transformed into E. coli BL21were induced by IPTG to express the E6 and E7 proteins. We used the SDS-PAGE to test the E6 and E7 proteins.Results:1. Using PCR technique to amplify extracted DNA of cervical cancer tissue sample as template, we obtained approximately 490bp and 310bp gene fragments;2. DNA sequencing results of the recombinant cloning plasmids pMD 19-T-E6 and pMD 19-T-E7 and expressing plasmids pET28a(+)-E6 and pET28a(+)-E7 showed that E6 gene mutated at the 253 base C253AT to T253AT, amino acid histidine changed into tyrosine, and E7 gene sequencing results showed the right of HPV16 E7 sequence;3. HPV16 E6, E7 protein were expressed by the plasmid pET28a (+)-E6, pET28a (+)-E7 in E.coli BL21.Conclusions:1. Successfully amplified the HPV16 E6 and E7 genes;2. Successfully constructed the prokaryotic expression plasmid of HPV16 E6 and E7 genes;3. Successfully induced the prokaryotic expression of HPV16 E6 and E7 proteins.