摘要
目的克隆人肝细胞生长因子（humanhepatocytegrowthfactor，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hHGFcDNA；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．coliDH5a细胞，鉴定pcDNA3．1＋-hH—GF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-致。②重组质粒pcDNA3．1＋-hHGF是具有表达功能的真核表达质粒。③pcDNA3．1＋-hHGF转染293T细胞，于转染后12、24、48、72h均能检测到hH-GF表达。结论成功构建hHGF真核表达载体，获得分泌性hHGF蛋白。
Objective To clone the gene encoding of human hepatocyte growth factor （ hHGF）, construct the eukary- otic 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 ＋ plas- mid 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 de- tected 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 ex- pression of hHGF could be detected, at 12, 24, 48 and 72 hours after pcDNA3.1 ＋ - hHGF transfected 293T cells. Conclu- sion hHGF eukaryotic expression vector is successfully constructed and the secreted hHGF protein is obtained.
出处
《徐州医学院学报》
CAS
2012年第12期803-806,共4页
Acta Academiae Medicinae Xuzhou
基金
基金项目：国家自然科学基金（30971281）
江苏省科教兴卫工程项目（XK2007025）
关键词
人肝细胞生长因子
TA克隆
亚克隆
真核表达载体
human hepatocyte growth factor
TA cloning
subcloning
eukaryotic expression vector
