低氧诱导因子-1α和角质细胞生长因子双基因重组减毒沙门菌菌株的构建及其在肠上皮细胞中的表达

Construction and Expression in Intestinal Epithelial Cells of a Recombinant Attenuated Salmonella Typhimurium Containing Hypoxia Inducible Factor 1α Gene and Human Keratinocyte Growth Factor Gene

目的构建同时携带低氧诱导因子-1α(HIF-1α)和角质细胞生长因子(KGF)真核表达载体的减毒沙门菌菌株(Ty21a-pIRES-HIF-IRES-KGF,TPHK),观察其在防治肠黏膜损伤方面潜在的应用前景。方法采用RT-PCR法从低氧处理A549细胞扩增HIF-1αcDNA后连接到载体pIRES-SEQ的NheI和MluI酶切位点,构建单基因重组质粒pIRES-HIF。然后以质粒pIRES2-EGFP-KGF为模板扩增KGF基因并克隆到重组质粒pIRES-HIF的XbaI和NotI酶切位点上,获得双基因重组质粒pIRES-HIF-IRES-KGF。采用电穿孔法将pIRES-HIF-IRES-KGF质粒转入减毒沙门菌Ty21a中,通过筛选获得TPHK。该菌株转染肠上皮细胞IEC-6后48 h,用ELISA法检测上清中HIF1α和KGF的表达水平。并采用MTT方法分析不同剂量表达产物对IEC-6细胞的作用。结果通过对构建质粒克隆进行测序及酶切,证实TPHK构建成功。TPHK转染正常肠上皮细胞IEC-6后,48 h取上清用ELISA法检测HIF和KGF的表达,结果表明6×105个细胞可表达(16.03±1.47)ng的HIF蛋白和(17.77±1.83)ng的KGF蛋白。MTT结果表明表达上清有明显刺激正常肠上皮细胞IEC-6增殖的活性(P<0.05),加入20%表达上清时刺激活性达高峰。结论成功构建了TPHK,其可有效转染肠上皮细胞IEC-6,表达上清可显著促进IEC-6细胞增殖。提示TPHK具有潜在的在肠黏膜损伤局部应用的前景。

Objective To construct a recombinant attenuated salmonella typhimurium expression vector （Ty21 a- pIRES-HIF-IRES-KGF, TPHK） simuhaneously containing hypoxia inducible factor 1αgene （HIF-1 α） and human kera- tinocyte growth factor gene （KGF） , and to investigate its potential application in prevention of intestinal mucomembra- nous injury. Methods HIF-1α gene was obtained from human lung cancer ceil line A549 which was cultured in hypoxia condition by RT-PCR method, and was subcloned into pIRES-SEQ vector at NheI and MluI sites （pIRES-HIF）. And then the KGF gene from plasmid pIRES2-EGFP-KGF by PCR was also subcloned into the plasmid pIRES-HIF at XbaI and NotI sites, and eukaryotic expression vector plRES-HIF-IRES-KGF was obtained. After being identified with restriction enzymes, plasmid pIRES-HIF-IRES-KGF was transformed into attenuated salmonella typhimurium strain Ty21a by elec- troporation method and directly transfected into intestinal epithelial cell IEC-6. The TPHK was obtained by screening. And the expression level of HIF-1α protein and KGF protein were detected at 48 h after transfection by enzyme linked im- munosorbent assay （ELISA）. The biological effects of the HIFledKGF-expressing product in different doses on intestinal epithelial cells were assessed by MTT colorimetric method. Results The recombinant of TPHK was successfully con- structed. The expression amount of HIF-1 protein and KGF protein was （ 16.03 ± 1.47） ng/6 × 10^5 cells and （ 17.77± 1.83） ng/6 × 10^5 cells in superuatant of intestinal epithelial cell IEC-6 at 48h after transfection respectively detected by ELISA. MTT results showed that the HIF-1/KGF-expressing product could significantly stimulate proliferation of intestinal epithelial cell IEC-6 （P 〈0. 05） , and the peak of stimulative activity appeared when 20% expressing product was add- ed. Conclusion A recombinant of TPHK can be constructed in vitro and express successfully in IEC-6 cells. It providesthe material basis for studying the biologic effect and potential application of the TPHK for intestinal mucosal injury.