人NT-proBNP的克隆及原核表达

目的：克隆人氨基末端脑钠肽（amino—terminal pro—brain natriuretic peptide，NT—proBNP）基因，构建原核表达载体并纯化表达产物。方法：采用PCR从正常成人cDNA中扩增NT—proBNP基因，将其克隆至pGEM—T中，测定其核苷酸序列。然后构建原核表达载体pGXE4T-2-NT-proBNP，用IPTG诱导表达，GSH—agarose亲和纯化蛋白。结果：经PCR扩增获得NT—proBNP基因，测序正确，在大肠杆菌中融合表达后，该蛋白的表达量占菌体总蛋白的20％，用SDS—PAGE和Western blot鉴定，显示其相对分子量34600。经亲和纯化后的GST—NT—proBNP的纯度可以达到96％，得率为1．8mg／100ml。结论：NT—proBNP的纯化成功，为建立NT—proBNP检测方法奠定了基础。

Cloning and Prokaryotic Expression of Recombinant Human Amino-Terminal Pro-Brain Natriuretic Peptide

Objective To clone human amino-terminal pro-brain natriuretic peptide gene and obtain purified NT-proBNP for future proparation of antibody.Methods With PCR technique,the gene encoding human amino-terminal pro-brain natriuretic peptide was cloned from the cDNA of human heart.Then this gene was inserted into expression vector pGXE4T-2.The constructed plasmid was expressed in E.Coli through IPTG induction and the expression product was purified by affinity chromatography through GSH-agarose.Results The acquired gene was expressed at 228bp apon electrophoresis and its sequence was correct.The PCR product was expressed in E coli BL21(DE3) with a high level as soluble protein,accounting for 20% of the total bacterial proteins.The gene product,characterized by SDS-PAGE and Western blot,appeared to be a fusion protein with molecular weight of 34600.After affinity chromatography,purity of the protein was over 96%.Conclusion The cloning prokaryotic expression and purificationg of human amino-terminal pro-brain natriuretic peptide would lead to the development of a quick diagnostic kit capable of clinical detectin of amino-terminal pro-brain natriuretic peptide.