【摘要】 目的系统地研究TREM-1的生物学功能,克隆TREM-1的cDNA,构建原核表达载体后,使其在大肠杆菌中得到表达。方法用RT-PCR从临床患者外周血白细胞中扩增TREM-1 cDNA,将其克隆到克隆载体pGEM-3Z上。经酶切及PCR扩增鉴定、测序后,再克隆到原核表达载体pT7-PL上,转化大肠杆菌BL21(DE3 plys),以IPTG诱导大肠杆菌表达带有氨酸和人TREM-1蛋白组成的融合蛋白,经SDS-聚丙烯酰胺凝胶(PAGE)电泳初步鉴定,并对融合蛋白进行纯化。结果酶切电泳和DNA测序结果表明,成功地克隆了人TREM-1 eDNA;SDS-PAGE电泳结果显示相应分子质量37ku处有含人TREM-1融合蛋白的初步表达,证明了TREM-1原核表达的可行性。更多还原

【Abstract】 Objective To clone TREM-1 cDNA, and to investigate the feasibility of prokaryotic expression of TREM-1, peripheral blood of trauma patients and healthy volunteers were collected. Methods TREM-1 cDNA was amplified by RT-PCR, cloned into vector pGZM-3Z, then amplified by PCR, authenticated by digestive enzymes, sequenced, constructed into expression vector pT7-PL, and expressed in E. coll. E. coli was induced by IPTG to express fusion protein composed of human TREM-1 protein and six lysines. The product was identified by using SDS-PAGE and purified. Results Authentication, PCR and DNA sequencing showed that the recombinant plasmid of human TREM-1 was successfully constructed. SDS-PAGE showed that human TREM-1 chimeric protein was successfully expressed in E. coli. Conclusion Our study showed that it was feasible to express TREM-1 in E. coli. Cloning of TREM-1 cDNA and its expression in E. coli facilitated determination of its biological function and further investigation of its roles in inflammation.更多还原