B型钠尿肽基因的克隆和原核表达载体构建及纯化  

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

作　　者：李卫鹏[1] 郑佐娅[1] 王红[1] 王从珠[1] 杭勤[1] 赵卫国[1] 

机构地区：[1]上海交通大学医学院上海市医学检验重点试验室,上海200023

出　　处：《蚌埠医学院学报》2007年第1期14-17,共4页Journal of Bengbu Medical College

摘　　要：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96 bp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W estern b lot鉴定大肠埃希菌中的表达产物,显示其相对分子量29 500。经亲和纯化后GST-BNP的纯度可以达到95%,500 m l菌液中得到纯化蛋白9.6 mg。多克隆抗体的效价为1∶32 000。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。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 pGXFAT-2. The construct pGXFAT-2/ BNP was expressed in E. eoli 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.

关 键 词：基因文库 人B型钠尿肽 克隆 分子 构建 纯化 

分 类 号：Q785[生物学—分子生物学]