人载脂蛋白M的表达与纯化

Expression and purification of human apolipoprotein M

目的:表达人细胞外全长结构域重组人载脂蛋白M(apolipoprotein M,ApoM)并将其纯化。方法:利用人类肝脏cDNA文库做模板,聚合酶链反应(PCR)法扩增ApoM DNA,将测序正确的目的基因片段插入质粒pGEXT相应位点,插入E.coli JM109,转化E.coli DL21(DE3),IPTG诱导蛋白表达。结果:PCR扩增产物经琼脂糖凝胶电泳证实在SDS-PAGE上出现一条560bp的基因片段。测序结果与GenBank公布的人ApoM基因序列完全一致。ApoM cDNA基因片段经IPTG诱导表达重组蛋白,SDS-PAGE电泳分析表明在相对分子量24kD左右出现新的蛋白条带。结论:成功克隆出人ApoM基因,重组表达出ApoM蛋白。

Objective To express and purify the extra cellular full-length human apolipoprotein M （ApoM）. Methods The ApoM gene fragment was amplified from the human liver cDNA library by PCR. The resulting product was cloned into pGEXT vector and sequenced. Then the confirmed canstatin cDNA was cloned into plasmid E. coli JM109 and then transformed into E. coli DL21 （DE3） where it was induced to express protein by IPTG. Results The ApoM gene was cloned by PCR and a 560 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoM gene in GenBank. Then ApoM cDNA gene fragment was induced by IPTG, and a 24 kD recombinant ApoM protein was tested on SDS-PAGE. Conclusion Human ApoM gene is successfully cloned and its recombinant proteins are expressed.