氨基末端B型钠尿肽基因的克隆和原核表达载体的构建及纯化

目的克隆人氨基末端B型钠尿肽(NT-proBNP)基因,构建原核表达载体并纯化其表达蛋白。方法用聚合酶链反应(PCR)从正常成人cDNA库中扩增出人NT-proBNP基因,将其克隆进pUCm-T中测定核苷酸序列。构建大肠杆菌分泌性表达载体pGEX-4T-3-NT-proBNP,IPTG诱导表达,GSH-agarose亲和纯化该表达蛋白。结果经PCR扩增成功获得228bp的NT-proBNP基因,测序正确,在大肠埃希菌中融合表达后,该蛋白的表达量占菌体总蛋白的23%,用SDS-PAGE和Westernblot鉴定大肠埃希菌中的表达产物,显示其相对分子质量为34600。经亲和纯化后的GST-NT-proBNP的纯度可以达到95%,得率为1.7mg/100ml。结论NT-proBNP基因的克隆、表达和纯化成功,为建立NT-proBNP检测方法奠定了基础。

Cloning, construction and purification of recombinant human amino-terminal pro-B type natriuretic peptide

Objective To clone human amino-terminal pro-B type natriuretic peptide (NT-proBNP) gene and purify its expression. Methods By using polymerase chain reaction (PCR) the gene encoding human NT-proBNP was cloned from the cDNA library and sequenced. Then this gene was inserted into expression vector pGEX-4T-3. The construct was expressed in Escherichia coli(E.coli) and the expression was purified by affinity chromatography through GSH-agarose. Results The acquired gene was 228 bp and its sequence was correct. The construct was expressed in E.coli with a high level as soluble protein, accounting for 23% of the total bacterial proteins. The gene product, characterized by SDS-PAGE and Western blot appeared to be a fusion protein with molecular mass of (34 600). The purity of the protein purified by affinity chromatography reached more than 95%. Conclusions The clone, expression and purification of human NT-proBNP lay a foundation of the development of diagnostic method for the detection of NT-proBNP.