关键词: 三氯乙烯, 癌蛋白SET, 重叠PCR, 串联亲和纯化, 融合表达

Abstract: BACKGROUND AND AIM: To construct the vector pcDNA3.1/SET-TAP carrying tandem affinity purification tag for the differentially expressed protein SET which can be used to further study protein-protein interactions of SET in L-02 liver cells. MATERIALS AND METHODS: Total RNA was extracted from L-02 liver cells: the open reading frame of SET was isolated by using RT-PCR and the TAP gene was amplified from plasmid.Adopting overlap PCR to construct the fusion gene(SET-TAP) through a chimeric primer,then the fusion gene and pcDNA3.1/zeo(+) were digested by EcoRⅠand XhoⅠ.The recombinant vector pcDNA3.1/SET-TAP was constructed through T4 ligase for 16 h at 16 ℃:and then transformed into E.coli DH 5α.After the sequence was confirmed by using double enzyme digestion and sequence analysis:L-02 liver cells were transiently transfected with recombinant vector via Lipofectamine 2000.The expression of fusion protein was preliminary detected by using real-time PCR and western blotting. RESULTS: Results from double enzyme digestion and sequencing showed that the fusion gene was correctly inserted into the vector pcDNA3.1/zeo(+).The pcDNA3.1/SET-TAP could transcribe and express the fusion protein effciently in L-02 liver cells verified by real-time PCR and western blotting. CONCLUSION: The recombinant vector pcDNA3.1/SET-TAP was successfully constructed,laying the foundation to further study the protein-protein interactions of oncoprotein SET in L-02 liver cells treated with trichloroethylene.