Construction of recombinant human angiostatin expression vector and stable expression on human hepatocelluar cancer cells

    TAO Kaiª²Shan, DOU Keª²Feng

    Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant human angiostatin (hANG) expression vector and to detect its expression in hepatocellular carcinoma cells. METHODS: hANG cDNA fragment was obtained by RTª²PCR from human embryonic tissue and the fragment was cloned into pCRª²Blunt IIª²TOPO vector and into pcDNA3.1 (+) expression vector. Colony PCR, restriction enzyme digestion and sequencing were used to verify the obtained hANG cDNA fragment. The recombinant pcDNA3.1ª²hANG plasmid was then transfected into hepatocellular carcinoma cells to detect the expression of hANG in these cells. RESULTS: The obtained hANG fragment (1.1 kb) was identical to the sequence of reported human angiostatin. Restriction enzyme digestion demonstrated that the recombinant pcDNA3.1ª²hANG expression vector was successfully constructed. The transfected SMMCª²7221 cells successfully expressed hANG. In addition, no difference in the growth speed was observed between the untransfected SMMCª²7221 cells, the SMMCª²7221 cells transfected with pcDNA3.1(+) vector and the SMMCª²7211 cells transfected with recombinant pcDNA3.1ª²hANG. CONCLUSION: We have successfully constructed the recombinant pcDNA3.1ª²hANG expression vector and obtained SMMCª²7221 cells that can stable express hANG. ......