B型钠尿肽基因的克隆和原核表达载体构建及纯化

目的: 克隆人B型钠尿肽(脑钠素,B-type natriuretic peptide,BNP)基因,纯化其表达蛋白并制备多克隆抗体。方法: 用PCR技术从正常成人心脏组织cDNA库中扩增出人BNP基因,将其克隆进pUCm-T中并测定核苷酸序列。构建大肠埃希菌分泌性表达载体pGXE4T-2/BNP,用异丙基β硫代半乳糖苷(IPTG)诱导表达,GSH-agarose亲和纯化表达蛋白,用纯化蛋白免疫BALB/c小鼠,制备多克隆抗体。结果: 经PCR扩增成功获得96bp的BNP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19%,用SDS-PAGE和Westernblot鉴定大肠埃希菌中的表达产物,显示其相对分子量29500。经亲和纯化后GST-BNP的纯度可以达到95%,500ml菌液中得到纯化蛋白9.6mg。多克隆抗体的效价为1:32000。结论: BNP基因的克隆、表达和纯化成功以及多抗的获得,为建立BNP检测方法奠定了基础。

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

Objective: To clone human B-type natriuretic peptide gene,purify the expressed protein and prepare its polyclonal antibodies.Methods: By using PCR technique,the gene encoding human B-type natriuretic peptide(BNP) was amplified from the cDNA library of human heart and sequenced.Then this gene was inserted into expression vector pGXE4T-2.The construct pGXE4T-2/BNP was expressed in E.coli and the expressed protein was purified by affinity chromatography through GSH-agarose.The purified protein was used to immunize BALB/c mice.Results: The cloned gene was 96 bp in length and its sequence was correct.The construct was expressed in E.coli with a high level of the protein as form soluble,accounting for 19% of the total bacterial proteins.The gene product,characterized by SDS-PAGE and Western blot appeared to be a protein with molecular mass of 29 500.The purity of the protein purified by affinity chromatography reached more than 95%.The titer of anti-sera was 1:32 000 after the fourth immunization.Conclusions: The success of gene clone,expression and purification of human BNP lay the foundation for developing a quick diagnostic kit applying to detecting BNP.