携带肝细胞生长因子基因的减毒沙门菌工程菌株的构建及其活性

Construction and Activity of Attenuated Salmonella Strain Carrying Hepatocyte Growth Factor Gene

目的构建携带肝细胞生长因子(Hepatocyte growth factor,HGF)基因的减毒沙门菌工程菌株,并检测其体外活性。方法从人胎盘cDNA文库中PCR扩增人HGF基因,克隆至真核表达载体pcK上,构建重组表达质粒pcKH,电穿孔法转入减毒沙门菌Ty21a中,获得携带HGF基因的减毒沙门菌工程菌株。将该菌株转染胃黏膜上皮细胞GES-1,48 h后采用ELISA方法检测上清中HGF蛋白的表达水平,MTT法检测不同浓度HGF表达产物对GES-1细胞增殖活力的影响。结果克隆的人HGF基因序列与GenBank中登录的人HGF cDNA序列(M60718.1)完全一致;重组表达质粒pcKH经双酶切鉴定构建正确;成功构建了携带HGF基因的减毒沙门菌工程菌株,可有效转染GES-1细胞,并表达活性HGF蛋白(14～16 ng/6×105个细胞),其可显著刺激GES-1细胞增殖(P<0.05)。结论已成功构建携带HGF基因的减毒沙门菌工程菌株,其可能具有在体内治疗胃肠疾病的潜能。

Objective To construct an attenuated salmonella strain carrying hepatocyte growth factor（HGF） gene and determine its activity in vitro.Methods Human HGF gene was amplified from human placental cDNA library by PCR and cloned into eukaryotic expression vector pcK.The constructed recombinant plasmid pcKH was transformed to attenuated Salmonella Ty21a by electroporation.Gastric mucosal epithelial cells GES-1 were transfected with the obtained attenuated Salmonella strain carrying HGF gene and,48 h later,determined for expression level of HGF protein in supernatant by ELISA,and analyzed for effect of HGF at various concentrations on proliferative activity of GES-1 cells by MTT method.Results The sequence of cloned human HGF gene was completely consistent with that of human HGF cDNA reported in GenBank（M60718.1）.Restriction analysis proved that recombinant plasmid pcKH was constructed correctly.Recombinant Salmonella strain carrying HGF gene was successfully constructed,which was tranfected into GES-1 cells effectively.Active HGF protein was expressed（14 ～ 16 ng / 6 × 105 cells）,which stimulated the proliferation of GES-1 cells significantly（P 0.05）.Conclusion An attenuated Salmonella strain carrying HGF gene was successfully constructed,with potential in treatment of gastrointestinal diseases in vivo.