人巨噬细胞金属弹力酶催化域基因的克隆及表达

Cloning and prokaryotic expression of human macrophage metalloelastase catalytic domain gene

目的克隆人巨噬细胞金属弹力酶催化域(HMEcd)基因,构建原核表达质粒,在大肠杆菌中表达HMEcd基因融合蛋白。方法用RT-PCR方法提取人胃癌组织总RNA并扩增出HMEcd cDNA,克隆入pMD18-T载体,构建克隆载体pMD18-T-HM Ecd,双酶切鉴定及DNA测序正确后再将HMEcd cDNA亚克隆至pET-28a(+)载体,构建原核表达载体pET-28a(+)-HM Ecd,转化大肠杆菌BL21(DE3)诱导表达,并行SDS-PAGE及Western blotting鉴定。结果RT-PCR获得预期的HMEcd cDNA,双酶切鉴定及DNA测序证实将HM Ecd基因分别正确插入克隆载体和原核表达载体中,SDS-PAGE及Western blotting鉴定证实表达出HMEcd融合蛋白。结论成功表达出HMEcd基因融合蛋白,为进一步功能实验研究奠定基础。

Objective To clone 507 bp cDNA fragment coding the human macrophage mctalloelastase catalytic domain and express it in E. coll. Methods HMEcd cDNA was amplified by reverse transcriptional polymerase chain reaction （RT-PCR） from tissue of gastric cancer. The fragment was inserted into pMDIS-T and then subcloned into pET-28a（ ＋ ） plasmid. The recombinant pMD18-T-HMEcd and pET-28a（ ＋ ）-HMEcd was identified by double digestion with restriction enzymes BamHⅠ and XhoⅠ and sequencing respectively, pET-28a（ ＋ ）-HMEcd was transformed into E. coll. BL21 （DE3）. Then the E. coil was inducede by IPTG. The fusion protein was analyzed by SDS-PAGE and identified by Western-blot. Results HMEcd cDNA was cloned. The pMD18-T-HMEcd and pET- 28a（ ＋ ）-HMEcd plasmid were conducted. SDS-PAGE and Western-blot demonstrated HMEcd protein was expressed in E. coli. Conclusions The recombinant HMEcd fusion protein is obtained successfully by prokaryotic expression,which establishas the foundation for successive studies.