重组质粒pIRES2-EGFP/CCK的构建及其在体内外的表达

目的构建猪源性胆囊收缩素(CCK)基因真核表达质粒pIRES2-EGFP/CCK,并在哺乳动物细胞COS-7中和仓鼠体内进行表达。方法从含CCK的中介载体pMD18-T/CCK中,以限制性内切酶方法获取目的片段,将其克隆入真核表达载体pIRES2-EGFP中,以脂质体法转染COS-7细胞,利用荧光显微镜观察绿色荧光蛋白的表达,同时采用肌肉注射法免疫仓鼠,了解重组质粒pIRES2-EGFP/CCK的体内表达。结果构建了猪CCK基因的重组真核表达质粒pIRES2-EGFP/CCK,用其转染COS-7细胞后24h、48h、72h均可检测到绿色荧光蛋白的表达,其中72h组荧光强度达到最强。用pIRES2-EGFP/CCK免疫仓鼠后,第4d注射部位可以检测到绿色荧光蛋白的表达,第14d荧光强度明显增强,第42d荧光强度进一步增强,正常对照组始终未检测到绿色荧光蛋白的表达。结论成功构建了猪源性CCK基因真核表达质粒pIRES2-EGFP/CCK,并成功地在哺乳动物细胞COS-7中和仓鼠体内进行了表达,为仓鼠胰腺癌的交叉免疫治疗奠定了基础。

Construction of Recombinant Plasmid pIRES2-EGFP/CCK and Its Expression in vivo and in vitro

Objective To construct eukaryotic expression plasmid of porcine CCK gene pIRES2-EGFP/CCK and express it in COS-7 cells and hamsters. Methods The aimed segments were obtained from intermediate vector pMD18-T/CCK by the method of restricted enzymatic resection and were inserted into a eukaryotic expression plasmid pIRES2-EGFP to construct a recombinant expression plasmid pIRES2-EGFP/CCK. The recombinant expression plasmid was transfected into COS-7 cells by liposome-mediated gene transfer method and observed through Fluorescence microscopy. The plasmid was injected into the skeletal muscle of hamsters directly to detect the expression of the recombinant plasmid in vivo. Results A recombinant eukaryotic expression plasmid pIRES2-EGFP/CCK was successfully constructed. Green fluorescent protein could be detected in the transfected COS-7 cells 24, 48, and 72 hours post transfection and the expression of green fluorescent protein reached its peak 72 h post transfection. The green fluorescent protein could be detected at the injection site on the 4th day post injection and the fluorescence intensity became stronger on the 14th day. The level of fluorescence became ever stronger on the 42nd day. No expression of green fluorescence was detected in the control group. Conclusion Porcine CCK cDNA eukaryotic expression plasmid pIRES2-EGFP/CCK has been successfully constructed and expressed in mammal cells COS-7 and hamster in vivo. The research paved the way for cross immunity therapy of hamster pancreatic carcinoma.