重组人可溶性THANK在大肠杆菌中的高效表达

目的:克隆人THANK的全长基因及编码可溶性胞外区的基因 ,并在大肠杆菌中表达可溶性THANK.方法:采用RT-PCR从PMA诱导的 HL60细胞中克隆人THANK全长编码基因,继以PCR扩增出可溶性THANK编码区基因(134～285位氨基酸),PCR产物克隆于pMD-18T,经DNA测序证实后亚克隆到pET-11a中.重组质粒转化B L21,以1 mmol/L IPTG进行诱导表达产物,SDS-PAGE分析表达产物,对重组蛋白经纯化复性后测定生物学活性.结果:RT-PCR扩增出编码人THANK全长编码基因及P CR扩增出THANK134-285基因的cDNA,经序列分析证实与文献报道完全一致.含有THA NK134-285编码基因的表达载体,转化大肠杆菌后表达出相对分子质量约18 000的重组蛋白.该重组蛋白经纯化后能够诱导U937细胞凋亡.结论:成功克隆了人THANK基因,并在大肠杆菌中表达重组可溶性THANK,该重组蛋白在实验条件下具有诱导U937细胞凋亡的作用.

High level expression of human THANK extracellular domain in E.coli

Objective:To clone and express human THANK extracellular dom ain(THANK1 34 - 2 85) in E.coli.Methods: U sing RT- PCR on total RNA from HL6 0 induced with PMA,hum an THANK gene was amplified and cloned into p MD- 18T and was sequenced.THANK1 34 - 2 85gene amplified from hum an THANK gene was also cloned and sequenced.THANK1 34 - 2 85 gene were subcloned into p ET- 11a and m ade a p ET- THANK1 34 - 2 85.BL2 1transformed with p ET- THANK1 34 - 2 85was used to analyze the expression of THANK1 34 - 2 85after induction with1mm ol/L IPTG.The recombinant THANK1 34 - 2 85was purified and its biological activity was analyzed on U937cell.Results:A4 6 8bp THANK1 34 - 2 85DNA fragm ent was cloned and sequenced. A p ET- THANK1 34 - 2 85was used to express recom binant THANK1 34 - 2 85.High level of THANK- ECD1 34 - 2 85expression(>4 0 % of total bacterial protein) was detected in BL2 1after induction with1mm ol/L IPTG.The recom binant protein was purified and detected in trigering U937apoptosis.Conclusion:THANK1 34 - 2 85gene is successfully cloned and highly expressed in E.coli,it shows apoptotic bioactivity on U937. [