肿瘤坏死因子-α转换酶基因金属蛋白酶区的克隆及表达

Cloning and expression of gene encoding metalloproteinase domain of tumor necrosis factor-α converting enzyme

目的:克隆和构建含有肿瘤坏死因子-α转换酶金属蛋白酶区(m etalloprote inase dom ain of hum an tumor necrosis factor-αconverting enzym e,TACEmp)基因表达载体,并在大肠埃希菌中高效表达。方法:用RT-PCR方法,从THP-1细胞中扩增出TACEmp基因片段,插入表达载体pET-D sbAmut,用限制性酶切和DNA测序进行鉴定;转化到大肠埃希菌BL21中进行诱导表达,通过SDS-PAGE电泳分析表达产物。结果:克隆TACEmp基因并构建重组表达载体pET-D sbAmut-TACEmp转化入BL21,IPTG诱导后获得高效表达的TACEmp融合表达蛋白。结论:利用分子伴侣融合蛋白技术使TACEmp在原核表达系统中获得了高效可溶性表达。

Objective：To construct expression vector of the metalloproteinase domain of human tumor necrosis factor-α converting enzyme （TACEmp） and express its protein in E-coll. Methods：RT-PCR and sequencing were used to clone and confirm the TACEmp gene. The fusion expression vector was constructed with the prokaryotic expression vector including disulfide isomerase dsbA gene and the TACEmp, named pET-DsbA^mut-TACEmp, and transformed into E. coli. BL21. After induced by IPTG, expression product was analyzed by SDS-PAGE. Results：TACEmp was cloned by RT-PCR. The recombinant vector pET-DsbA^mut-TACEmp was constructed. The fusion protein was expressed at high level in E. coll. Conclusions： The TACEmp was expressed at high level in prokaryotic expression system by using fusion protein technology. The expression of TACEmp protein may be useful for the study of biological funetions of TACEmp and it＇ s biotherapy in TACEmp related diseases.