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目的为研究三氯乙烯诱导的差异蛋白SET在肝细胞L-02中的相互作用,构建了癌蛋白SET和His标签融合表达的真核表达载体pcDNA3.1(+)/SET-His。方法从L-02肝细胞中提取总RNA,采用RT-PCR扩增SET-His基因并进行双酶切,序列纯化后定向克隆至pcDNA3.1/zeo(+)载体,阳性克隆载体进行双酶切和测序鉴定,阳性克隆载体瞬时转染入L-02肝细胞,利用Western blotting检测SET-His融合蛋白的表达。结果利用RT-PCR从L-02细胞总RNA中成功克隆出SET基因,经双酶切和测序鉴定证实pcDNA3.1(+)/SET-His真核表达载体构建成功。经Western blotting验证表明,SET-His融合蛋白在肝细胞中获得高效表达。结论该结果为研究SET蛋白相互作用以及三氯乙烯致机体损伤的机理奠定了基础。
OBJECTIVE: To study the interaction of SET induced by Trichlorethylene in hepatocyte L-02, an eukaryotic expression vector pcDNA3.1 (+) / SET-His with fusion protein SET and His tag was constructed. METHODS: The total RNA was extracted from L-02 hepatocytes. The SET-His gene was amplified by RT-PCR and double digested. The sequence was purified and cloned into pcDNA3.1 / zeo (+) vector. The positive clones were transiently transfected into L-02 hepatocytes and the expression of SET-His fusion protein was detected by Western blotting. Results The SET gene was successfully cloned from total RNA of L-02 cells by RT-PCR. The double-digestion and sequencing confirmed that the eukaryotic expression vector pcDNA3.1 (+) / SET-His was successfully constructed. Western blotting showed that SET-His fusion protein was highly expressed in hepatocytes. Conclusion The results provide the basis for studying the interaction of SET protein and the mechanism of trichlorethylene-induced injury.