摘要
目的 胆囊收缩素 (Cholecystokinin,CCK)在胰腺癌的发生发展中起着很重要得作用 ,本实验的目的是构建猪源性 CCK基因真核表达质粒 p IRES2 - 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 p MD18- 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 p IRES2 - 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 p IRES2 - 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 p IRES2 - EGFP/ CCK,用其转染 COS- 7细胞后 2 4 h、4 8h、72 h均可检测到绿色荧光蛋白的表达 ,其中 72 h组荧光强度达到最强。用 p IRES2 - EGFP/ CCK免疫仓鼠后 ,第 4天注射部位可以检测到绿色荧光蛋白的表达 ,第 14天荧光强度明显增强 ,第 4 2天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 p IRES2 - 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 。
Objective AIM Cholecystokinin(CCK) plays an important role in the generation and progression of pancreatic carcinoma. The research aims 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 72h 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. The control group was never detected of the expression of green fluorescence.Conclusion Porcine CCK cDNA eukaryotic expression plasmid pIRES2-EGFP/CCK has been constructed successfully, and was expressed in mammal cells COS-7 and hamster in vivo. The research paved the way for cross immunity therapy of hamster pancreatic carcinoma.
出处
《肝胆外科杂志》
2005年第1期63-65,共3页
Journal of Hepatobiliary Surgery
基金
国家自然科学基金资助项目 (基金编号 3 0 170 917)
