【作者】 鲍德明；

【导师】 胡乃中；

【作者基本信息】 安徽医科大学 ， 内科学， 2007， 硕士

【摘要】 目的本研究通过构建人巨噬细胞金属弹力酶催化域(HMEcd)基因原核表达质粒,诱导大肠杆菌表达融合6×His tag的HMEcd基因融合蛋白,经纯化、复性后用于HME体内外干预胃癌细胞实验研究。方法提取手术切除的新鲜胃癌组织总RNA,逆转录合成cDNA第一链,设计一对特异性引物PCR扩增HMEcd cDNA,1%琼脂糖凝胶电泳鉴定并切胶纯化后与pMD18-T载体连接,构建克隆质粒pMD18-T-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序正确后再将HMEcd cDNA亚克隆至pET-28a(+)载体,构建原核表达质粒pET-28a(+)-HMEcd,转化大肠杆菌DH5α,提取质粒,BamH I和Xho I双酶切鉴定及DNA测序后,转化大肠杆菌BL21(DE3)诱导表达,SDS-PAGE及Western blot鉴定。His Band镍柱亲和纯化,透析重折叠复性,浓缩后测定蛋白质浓度,明胶酶谱分析及I型胶原蛋白分解实验鉴定酶活性。结果RT-PCR获得预期的HMEcd cDNA,双酶切鉴定及DNA测序证实分别成功构建克隆质粒pMD18-T-HMEcd和原核表达质粒pET-28a(+)-HMEcd,SDS-PAGE及Western blot证实在大肠杆菌中诱导表达出HMEcd基因融合蛋白,诱导5h蛋白表达量占菌体总蛋白的18.92%。纯化后SDS-PAGE分析蛋白质纯度达98%以上。重折叠复性后经活性鉴定证实HMEcd基因融合蛋白具有分解明胶及I型胶原蛋白的酶活性。结论获得HMEcd cDNA,成功构建原核表达质粒;在大肠杆菌中高效表达出HMEcd基因融合蛋白;建立融合蛋白亲和纯化及体外重折叠复性方法,成功获得高纯度活性HMEcd基因融合蛋白;为下一步HME对胃癌细胞的体内外干预研究创造了条件。更多还原

【Abstract】 Objective In this experiment we cloned 507 bp cDNA fragment coding for human macrophage metalloelastase catalytic domain (HMEcd) and constructed a prokaryotic expression plasmid and expressed it in E. coli.Then HMEcd protein was purified and renatured, which can be used in the studies on tumor intervention with HME.Methods HMEcd cDNA was amplified by reverse transcriptional polymerase chain reaction(RT-PCR) from tissue of gastric cancer and identified by 1% agarose gel electrophoresis. The fragment was inserted into pMD18-T vector and constructed pMD18-T-HMEcd plasmid. The plasmid was transformed into E. coli DH5αand amplified in the bacterium. Then we extracted pMD18-T-HMEcd plasmid from E. coli DH5αand identified the recombinant plasmid by double digestion with restriction enzymes BamH I and Xho I and sequencing. HMEcd cDNA cutted from pMD18-T-HMEcd was subcloned into pET-28a(+) vector. The recombinant pET-28a(+)-HMEcd was identified with the same method for pMD18-T-HMEcd and transformed into E. coli BL21(DE3) and induced by IPTG. The production of expression was analyzed by SDS-PAGE and detected by Western-blot. The fusion protein was purified with His Bind Collumn and refolded by dialysis and assaied after concentration. The enzymatic activity of recombinant protein was confirmed by gelatin zymography and cleavage of type I collagen in vitro.Results HMEcd cDNA was cloned. The pMD18-T-HMEcd and pET-28a(+)-HMEcd plasmid were conducted and confirmed. SDS-PAGE and Western-blot demonstrated HMEcd protein was expressed in E. coli BL-21(DE3). The recombinant protein occupied approximately 18.92% of total bacterial protein. HMEcd protein was purified with a purity of over 98% and demonstrated the enzymatic activity.Conclusions We obtained HMEcd cDNA and prokaryotic expressive plasmid perfectly.The recombinant HMEcd fusion protein was expressed successfully. We established the purification and refolding scheme through the experiments. Which laid the foundation for successive studies.更多还原