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应用RT-PCR技术,从人白血病多药耐药细胞株K562/ADR中扩增出肿瘤坏死因子受体相关蛋白1(tumor nec-rosis factor receptor-associated protein1,TRAP1)基因的cDNA。选择NdeⅠ和XhoⅠ分别作为上下游引物的酶切位点,将TRAP1基因克隆到带有6×His标签的pET-28a(+)的载体上。重组质粒转化大肠杆菌DH5α中,涂布于含卡那霉素的LB琼脂培养基上,经双酶切鉴定后的阳性克隆送去测序。测序成功的重组质粒pET28a(+)-TRAP1转化大肠杆菌BL21(DE3)中,在IPTG的诱导下,成功表达重组蛋白TRAP1。通过改变诱导温度、诱导时机、IPTG浓度及诱导时间,找出最佳表达条件,使重组蛋白TRAP1表达量最高。结果表明,在39℃条件下,OD600达到0.8时,经终浓度为0.1 mmol/L的IPTG诱导6 h,目的蛋白的表达量最高。该研究为纯化出TRAP1蛋白,进一步研究该蛋白的结构和功能奠定了基础。
The cDNA of tumor necrosis factor receptor-associated protein 1 (TRAP1) gene was amplified by RT-PCR from K562 / ADR human multidrug-resistant cell line. Select Nde Ⅰ and Xho Ⅰ as upstream and downstream primer cleavage sites, the TRAP1 gene was cloned into the 6 × His-tagged pET-28a (+) vector. Recombinant plasmids were transformed into E. coli DH5α, plated on kanamycin-containing LB agar medium, and the positive clones were identified by double enzyme digestion and sent to sequencing. The successfully sequenced recombinant plasmid pET28a (+) - TRAP1 was transformed into E. coli BL21 (DE3) and the recombinant protein TRAP1 was successfully expressed under the induction of IPTG. By changing the induction temperature, induction time, IPTG concentration and induction time, find out the best expression conditions, so that the highest expression of recombinant protein TRAP1. The results showed that under the conditions of 39 ℃ OD600 reached 0.8, the final protein was induced by IPTG at a final concentration of 0.1 mmol / L for 6 h. This study laid the foundation for the purification of TRAP1 protein and further study of its structure and function.