| [Objective] Interferon-γ (hIFN-γ) is a very important tumor immunity correlated cytokine, which can not only inhibit cell proliferation, adjust cell differentiation, restrain invasion and metastasis of hepatocellular carcinoma, but also has complicated interaction with many immunocytes and cytokines on molecule and gene lever, thus to enhance the immunologic function of anti-hepatocellular carcinoma. The hIFN-γ expression vector pcDNA4His/Max-hIFN-Y was constructed by inserting hIFN-γ gene into a recombinant plasmid vector pcDNA4His/Max, and investigated the inhibitory effects of cationic liposome-mediated gene transfection of hIFN-γ on hepatocellular carcinoma in vitro. [Methods] The primer pairs were designed according to gene sequence of human hIFN-γ mRNA in GenBank, inserting BamHI and EcoRI restriction site into the upstream and downstream primer respectively. The whole length cDNA of hIFN-γ was amplified from Peripheral blood of healthier by one step RT-PCR, and cloned into the eukaryotic expression vector pcDNA4His/Max. After restriction enzyme digest and gene sequencing, the recombinant expressing vector pcDNA4His/MaxC-hIFN-y was constructed correctly. Recombinant plasmid pcDNA4His/MaxB-EGFP was transfected into human hepatocellular carcinoma cell line HepG2 to determine the best transfection efficiency. The ultrapure recombinant plasmid containing hIFN-γ cDNA was prepared, and then transfected into HepG2 mediated by liposome. The expression of hIFN-γ were identified by ELISA. [ Results ] hIFN-γ mRNA was detected only in pcDNA4His/Max-hIFN-γ transfected cell lines, and high levels of hIFN-γ protein were detected only in the supernatants of hlFN-y gene transfected cell lines. [ Conclusion ]The recombinant eukaryotic expression plasmid pcDNA4His/Max-hIFN-γ was constructed successfully. The hIFN-γ gene can be effectively transfected into HepG2 mediated by liposome, and sustained expression was obtained.
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