重组人融合基因Nm23—H1／HbFGF的构建及其在大肠杆菌中表达？…

根据Ｎｍ２３－Ｈ１与ＨｂＦＧＦｃＤＮＡ序列，人工合成一段中间核酸序列，将它分别与Ｎｍ２３－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Ｎｍ２３－Ｈ１／ＨｂＦＧＦ，将其定向克隆于质粒载体ｐＢＶ２２０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

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

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 degrees C 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 34 kD. 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.