Construction Of Recombinant Adeno-Associated Virus Vector Expressing HSVtk And The Studies Of Human Breast Cancer Gene Therapy

Human breast cancer is a common malignancy in women all over the world. Operation, radiotherapy and chemotherapy are three patterns in the therapy of human tumor. With the development of molecular biology and biotechnology, biotherapy become more and more important and become the forth pattern in tumor gene therapy. Suicide gene therapy is one of the choices and a highlight in human gene therapy recently.In this research, the HSVtk gene, which is controlled by Tet gene regulation system, was inserted into AAV vector. Two viral vectors: AAV/TRE/HSVtk/Tet-On and AAV/TRE/HSVtk/Tet-Off were constructed. PCR amplification was performed using primers based on Tet-On/ Tet-Off gene sequence from pRevTet-On/pRevTet-Off. Tet-On/Tet-Off gene was inserted into pRevTRE/HSVtk and then the recombinant was digested by Xho I and Cla I. The fragment, which contains HSVtk gene and Tet-On/ Tet-Off gene, was inserted into pAAV-MCS. The PCA/K promoter, which is not controlled by Tet-On/ Tet-Off gene, was got rid of by partially digested. The end was flushed by Klenow DNA polymerase and then connected by T4 DNA ligase. pAAV/TRE/HSVtk/Tet-On or pAAV/TRE/HSVtk/Tet-On, pAAV-RC, pHelper were co-transfected into incasing cells HEK293 by using modified calcium phosphate co-precipitation method. Cells transfected with those vectors were harvested by scraping at 3 or 4 days after the initial transfection and lysed through three freeze/thaw cycles. Virus was purified by CsCl2 density gradient purification method. The liter of those viruses are 2.37X 10n/ml and 3.86X 10!Vml . The two recombinant AAV viruses were used to infect MCF-7 cell line. The HSVtk gene in MCF-7 genome was detected by dot-blot after the cells was infected by rAAV.In addition, transfection of pRevTRE/HSVtk/Tet-On was performed for MCF-7 cell line by using lipofectin. After selected by Hygromycin B, MCF-7 cell line stably expressing Tet-regulated HSVtk gene was established. mRNA level of MCF/RevTRE/HSVtk/Tet-On at different Dox concentration was detected by RT-PCR and MTT method was performed to detect the cell survival rate of MCF/TRE/HSVtk/Tet-On at different Dox concentration when GCVIVpresented. The product of HSVtk gene can convert the nontoxic GCV into toxic product and kill the breast cancer cells. The expression of HSVtk gene was controlled by Tet-On regulation system under the inducement of Dox.