人线粒体核糖体蛋白S17 cDNA克隆及其在大肠杆菌中的表达和纯化  

Cloning, expression and purification of human MRPS17 cDNA in Escherichia coli

作　　者：杨斌[1] 郝飞[1] 杨卫兵[1] 杨希川[1] 钟白玉[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038

出　　处：《解放军医学杂志》2006年第1期42-44,共3页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金资助项目(30300180);重庆市自然科学基金资助项目(CSTC2004BB5043);中华医学会欧来雅中国人健康皮肤研究项目(2005020641);第三军医大学科研基金资助项目(XG200314)

摘　　要：目的克隆人MRPS17cDNA,并在大肠杆菌中表达纯化。方法从人原代培养毛乳头细胞中分离总RNA,采用RTPCR及序列测定等方法获得人MRPS17cDNA,然后克隆至原核表达载体pET28a,转化至大肠杆菌BL21(DE3),IPTG诱导表达;C末端融合了6×His纯化标签的表达产物行Western印迹法分析并用Ni2+NTA离子交换树脂进行纯化;纯化蛋白行SDSPAGE纯度分析。结果获得了人MRPS17cDNA,与GenBank报道的序列一致,并成功构建了高效原核表达质粒pET28aMRPS17;Western印迹结果表明,经IPTG诱导,在大肠杆菌中表达了分子量约13kD的目的蛋白;表达产物经Ni2+NTA离子交换树脂纯化,纯度>90%。结论克隆得到了人MRPS17cDNA,并在E.coli中成功表达和纯化了MRPS17蛋白,为后续研究奠定了基础。Objective To clone, express and purificate human MRPS17 cDNA in Escherichia coil （E- coli）. Methods MRPS17 cDNA was obtained from total RNA isolated from primary cultured human hair papillary cells by RT PCR and sequencing method, and then it was inserted into prokaryotic expression vector pET28a for the IPTG-induced expression in E. coli BL2I （DE3）. The expression product fused with 6 × His at C terminal was analyzed by Western blotting, and purified by using Ni^2＋ -NTA ion exchange resin. The purity of MRPS17 protein was analyzed by SDS-PAGE. Results Human MILPS17 cDNA was obtained, and the expression plasmid pET28a- MRPS17 was constructed successfully. Western blot analysis showed that human MRPS17 with 13kD molecular weight was expressed in E. coli at 20℃. The purity of the recombinant MILPS17 was more than 90% after purification using Ni^2＋ -2NTA ion exchange resin. Conclusion The sequence of MRPS17 cDNA was consistent with the known sequence from the Genbank. MRPS17 is successfully induced and expressed in E. coli.

关 键 词：核糖体蛋白质类 DNA 互补 基因表达 

分 类 号：Q781[生物学—分子生物学]