【摘要】 目的 克隆人肝细胞生长因子(human hepatocyte growth factor,hHGF)的编码基因,构建真核表达载体,获得hHGF蛋白。方法 从含hHGF的人肝脏组织总RNA中,利用RT-PCR方法扩增出hHGF cDNA;利用TA克隆技术,将该基因片段克隆至真核表达载体pcDNA3.1~+质粒,转化E.coli DH5α细胞,鉴定pcDNA3.1~+-hHGF重组质粒。将重组质粒pcDNA3.1~+-hHGF转染293T细胞,应用ELISA方法检测hHGF在293T细胞中的表达。结果(1)测序结果证实本实验克隆的基因与人HGF基因序列一致。(2)重组质粒pcDNA3.1~+-hHCF是具有表达功能的真核表达质粒。(3)pcDNA3.1~+-hHGF转染293T细胞,于转染后12、24、48、72 h均能检测到hHGF表达。结论 成功构建hHGF真核表达载体,获得分泌性hHGF蛋白。更多还原

【Abstract】 Objective To clone the gene encoding of human hepatocyte growth factor(hHGF),construct the eukaryotic expression vector,and harvest hHGF protein.Methods hHGF cDNA was amplified by RT-PCR from total RNA of human liver tissue containing hHGF,the gene fragment was cloned into the eukaryotic expression vector pcDNA3.1~+ plasmid by TA cloning technique and then transformed E.coli DH5α cells,identification of recombinant plasmid pcDNA3.1~+-hHGF.Recombinant plasmid pcDNA3.1~+-hHGF transfected 293T cells.The expression of hHGF in 293T cells was detected by ELISA.Results(1)Sequencing results confirmed that the cloned gene was identical to the hHGF gene sequence.(2)The recombinant plasmid pcDNA3.1~+-hHGF was an expression of functional eukaryotic expression plasmid.(3)The expression of hHGF could be detected,at 12,24,48 and 72 hours after pcDNA3.1~+-hHGF transfected 293T cells.Conclusion hHGF eukaryotic expression vector is successfully constructed and the secreted hHGF protein is obtained.更多还原