人NT-proBNP的克隆及原核表达

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

目的:克隆人氨基末端脑钠肽(am ino-term inal 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W estern b lot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/100m l。结论:NT-proBNP的纯化成功,为建立NT-proBNP检测方法奠定了基础。

Objective To clone human amino - terminal pro - brain natriuretic peptide gene and obtain purified NT - proBNP for future preparation of antibody. Methods With PCR technique, the gene encoding human amino - terminal pm- brain natriuretic peptide was cloned from the cDNA of human heart. Then this gene was inserted into expression vector pGXEAT - 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 eoli 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 - ternlinal 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.