人肝细胞生长因子在CHO细胞中的表达

Expression of human hepatocyte growth factor in CHO cells

以ｐＲＣ／ＣＭＶ作为真核细胞表达性载体，用限制性内切酶ＢａｍＨＩ／ＸｂａⅠ将ＨＧＦｃＤＮＡ全序列定向重组到ｐＲＣ／ＣＭＶ的多克隆位点上。筛选得到的重组质粒以磷酸钙ＤＮＡ共沉淀的方法转染ＣＨＯ细胞，经８００ｍｇ／Ｌ的Ｇ４１８筛选，获得阳性细胞克隆。用ＨＣＣ－７９０２细胞及原代培养的成年大鼠肝细胞对转染阳性细胞培养基收集上清进行活性测定。３Ｈ－ＴｄＲ掺入等分析证明，它能有效地抑制体外培养的人肝癌细胞的生长，并可明显刺激鼠肝细胞ＤＮＡ的合成。结果表明，重组人ＨＧＦ基因在ＣＨＯ细胞中成功地获得表达。

pRC/CMV-HGF plasmid was constructed by recovering HGF cDNA fragment from pBSks-HGF with BamH Ⅰand XbaⅠdigestion ,and inserting it directly to pRC/CMV,a mammalian expression vector at the same sites(BamH Ⅰ/XbaⅠ).The analysis of multiple RE mapping indicated that the recombinant expression plasmid had correct cloning orientation and perfect structure.By the method of DNA-calcium phosphate co-precipitation,pRC/CMV-HGF were transfered into CHO cells.Screened with G418 at 800 mg/L,the transfected cell clones were obtained.Activity analysis by 3 H-TdR incorperation revealed that the activities as native HGF were detected in transfected cell medium,which can inhibit the growth of HCC-7902 and can stimulate the DNA synthesis in rat hepatocytes.All these results show that the mammalian expression system of HGF can be an effective source of providing HGF in vitro.