Construction And Analysis Of The Recombinant Retroviral Vector System With Human Tum-5 Gene

Objective: To construct recombinant retroviral vector carrying tum-5 gene which is the anti-angiogenesis activity fragment of human tumstatin gene, and package this recombinant retroviral vector into false virus particles with packaging cell PA317, and analyze it using NIH3T3 cells.Methods: Total RNA was extract from fetal kidney tissue with Trizol reagent. Tum-5 fragment was amplified by RT-PCR, and directionally cloned into the MCS of retroviral plasmid pLXSN,which then was transfected into PA317 cells with electroporation method.The positive PA317 cells clones were selected and amplified with G418. The fake virus particles with Tum-5 were collected and were infected NIH3T3 cells to detect the virus titer and safety.Results: Tum-5 gene contain 255 bp including restriction site was gained by RT-PCR method and was directionally cloned to pLXSN retroviral vector. the sequencing results show that the Tum-5 gene sequences was as same as the cloning and sequencing of the genes in Gene Bank provided by totally consistent.It was confirmed that there have specific Tum-5 fragments in the PA317 and NIH3T3 cells after transfection , the virus titer was 2.05×104cfu/ml. The supernatant of cultured of NIH3T3 after transfection has not infected the new NIH3T3 cells .Conclusions: Retroviral vector system carrying human Tum-5 gene was successful constructed, and its characters and effects were tested for creating a new, effective and safe way to transfect endogenous gene in gene therapy of anti-tumor angiogenesis and dependent vascular disease.