重组人融合基因Nm23-H1/HbFGF的构建及其在大肠杆菌中表达的研究  

Studies on the Construction and Expression of Recombinant Human Fusion Gene Nm23-H1/HbFGF in Escherichia coli

作　　者：孙艳梅[1] 赵利淦[1] 张美英[1] 

机构地区：[1]暨南大学生物工程研究所,广州510632

出　　处：《生物工程学报》2000年第5期551-556,共6页Chinese Journal of Biotechnology

基　　金：国务院侨办重点攻关项目资助

摘　　要：根据Nm2 3 H1与HbFGFcDNA序列 ,人工合成一段中间核酸序列 ,将它分别与Nm2 3 H1cDNA的上游引物及HbFGFcDNA的下游引物组成两对引物 ,通过PCR(聚合酶链式反应 )构建出融合基因Nm2 3 H1/HbFGF ,将其定向克隆于质粒载体 pBV2 2 0上 ,经诱导 ,SDS PAGE分析 ,表达产物分子量为 34kD ,表达量占菌体总蛋白的14% ,表达产物以包涵体形式存在 ,ELISA和Western印迹表明包涵体具有Nm2 3 H1和HbFGF抗原性 ,然后对包涵体进行变性、复性及纯化处理 ,取纯化产物进行定性和生物活性分析 ,结果证明纯化产物具有Nm2 3 H1和HbFGF的抗原性及生物活性。以上实验为今后进一步研究融合基因Nm2 3 H1/HbFGF在真核细胞中的抑癌、致癌性质打下了基础。First,a piece of intermediate nucleic acid chain was designed according to the nucleic acid sequences of Nm23\|H1 and HbFGF cDNA,then it was combined with the upstream primer of Nm23\|H1 or the downstream primer of HbFGF to perform Polymerase Chain Reaction (PCR) respectively.The fusion gene Nm23\|H1/HbFGF was constructed by four steps of PCR,and it was cloned into the plasmid vector pBV220.The recombinant was induced at 42℃ and analyzed by SDS\|PAGE.Results show that the fusion gene Nm23\|H1/HbFGF highly expresses its product in inclusion body in E.coli BL21(DE3).The expressing product is 14% of the total bacterial protein and it is 34kD.ELISA assay and Western blot indicate that the inclusion body contains antigens of Nm23\|H1 and HbFGF.By denaturation,renaturation and purification,the inclusion body was purified.Determination and biological activity assay show that the purified product contains two kinds of antigens which have biological activities of Nm23\|H1 and HbFGF.All these data establish good base to study the tumor suppressor activity and oncogenicity of the fusion gene Nm23\|H1/HbFGF in eukaryocyte.

关 键 词：融合基因Nm23-H1/HbFGF 生物活性 基因表达 

分 类 号：Q78[生物学—分子生物学]