IL-24融合蛋白表达载体的构建及其对人小肠癌细胞的生长抑制作用

Construction of IL-24 gene expression vector and inhibitory effect of IL-24 fusion protein on growth of human intestinal carcinoma cells

目的:构建人白细胞介素-24(IL-24)基因的表达载体并在大肠杆菌中表达．研究IL-24融合蛋白对人小肠癌细胞(HIC)的生长抑制作用．方法:用PCR从质粒TRAP-hIL-24中扩增人 IL-24 cDNA片段,并将该片段插入DGEX-KG 原核表达载体中,实现插入基因的融合表达．用SDS-PAGE和Western blot对表达产物进行鉴定．提取并纯化IL-24融合蛋白,以不同浓度与 HIC细胞共培养72 h,同时设立相应浓度梯度的GST蛋白组、HIC细胞对照组及空白孔,采用MTT法检测各组人小肠癌细胞的体外增殖结果:酶切结果证实,成功地构建了pGEX- KG-IL-24原核表达载体,并在大肠杆菌中获得稳定的表达,表达产物的相对分子质量(Mr) 同预期值相一致,融合蛋白为Mr53 000,GST 蛋白为Mr29 000．IL-24融合蛋白以浓度依赖性的关系抑制HIC细胞的生长,并且在20和40 mg／L时的抑制率明显高于GST蛋白．结论:成功地构建了重组表达载体pGEX-KG- IL-24,并在E.coli BL21中表达．IL-24融合蛋白对HIC细胞生长有抑制作用．

AIM： To construct a recombinant expression vector of human interleukin-24 （IL-24） gene and express it in E. coli, and to investigate the inhibitory effect of IL-24-GST fusion protein on the growth of human intestinal carcinoma （HIC） cells. METHODS： The human IL-24 cDNA fragment was amplified from plasmid TRAP-hIL-24 by polymerase chain reaction （PCR）, then cloned into the prokaryotic vector pGEX-KG, and expressed inductively as a fusion protein in E. coli. The expressed GST-IL-24 fusion protein was purified via GST-Sepharose 4B Column and identified by SDS-PAGE and Western blot. In vitro cultured human HIC cells were treated with different concentrations of GST-IL-24 fusion protein and GST protein, and the proliferation of HIC cells was measured by MTF assay. RESULTS： Restriction enzyme digestion analysis showed that the recombinant prokaryotic expression vector pGEX-KG-IL-24 was constructed successfully and expressed stably in E. coli. The relative molecular mass （Mr） of the expression products was 53 000（GST-IL-24） and 29 000（GST）, respectively, which was identical with the predictive value. GST-IL-24 fusion protein inhibited the proliferation of HIC cells in a concentration-dependent manner, and the inhibitory rates for 20 and 40 mg/L were obviously higher in fusion protein-treated cells than those in GST-treated cells. CONCLUSION： The recombinant expression vector pGEX-KG-IL-24 is constructed successfully and expressed as a bioactive fusion protein in E. coli. GST-IL-24 fusion protein inhibits the growth of HIC cells in a concentration-dependent manner.