Constructtion of the Recombinant pAAVCMVp53 for Cervical Cancer Gene therapy / 대한산부인과학회잡지

ABSTRACT

OBJECTIVE: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). METHODS: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. RESULT: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. CONCLUSION: This system will be one of the useful vector system for cervical cancer gene therapy.