[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.
Sun
Y M
YM
Bioengineering Institute of Jinan University, Guangzhou 510632.
Zhao
L G
LG
Zhang
M Y
MY
D004926:Escherichia coli / Q000235:genetics*; D016222:Fibroblast Growth Factor 2 / Q000235:genetics*; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics*; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D014157:Transcription Factors / Q000235:genetics*
D054778:NM23 Nucleoside Diphosphate Kinases; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D020559:Monomeric GTP-Binding Proteins