双基因表达载体的构建及生物活性观察  

Construction of Double-Gene Expression Vector and Its Biological Effects

作　　者：艾国平[1] 谭虎[1] 粟永萍[1] 王涛[1] 冉新泽[1] 

机构地区：[1]第三军医大学全军复合伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《现代生物医学进展》2008年第3期404-407,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金重点项目(30230360);面上项目资助(30470527)

摘　　要：为了促进肠上皮细胞增殖、加速肠上皮机械屏障修复和防止肠源性感染的发生,利用分子克隆技术首先获得含胰高血糖素样肽-2(GLP-2)编码序列的高血糖素原cDNA片段,通过PCR重叠延伸技术成功拼接GLP2的信号肽及成熟肽,同时将成熟肽N-末端第二位丙氨酸的序列(GCT)定点突变为GGT序列(甘氨酸);并以我室保存的pcDNA3.1-HD-5-cDNA为模板克隆防御素-5(HD-5)信号肽及成熟肽序列,分别构建到双基因表达载体pVITRO3,转染肠上皮细胞并观察其对肠上皮细胞增殖活性和杀菌能力的影响,结果显示构建的pVITRO3-HD-5-GLP2双基因性表达载体具有促进肠上皮细胞增殖和抑制细菌增殖和黏附的作用。The purpose is to promote proliferation of intestinal epithelial cells , accelerate the rehabilitation of intestinal mechanical barrier and prevent intestinal bacteria translocation. We cloned the proglucagon cDNA including GLP-2 gene sequence by polymerase chain reaction and reverse transcription. The signal peptides and mature peptides of GLP2 which were produced by PCR were linked by SOE （splicing by overlap extension） The second amino acid in the N-terminal of GLP-2, was mutated from GCT （coded alanine） to GGT （coded glycin） by PCR. The human gene defensin 5 was cloned from pcDNA3.1-HD-5-cDNA, then we constructed the GLP-2 and HD-5 cDNA into double-gene expression vector pVITRO3 and tranfected intestinal epithelium cells to observe the ability of proliferation and anti-bacterium of IEC-6 and Caco-2 cells. The results showed that construction of double gene expression vector had the ability of prompting proliferation of cells and inhibiting the multiplication and adherence of bacteria.

关 键 词：胰高血糖素样肽-2 防御素-5 双基因表达载体 肠上皮细胞增殖 细菌黏附 

分 类 号：Q78[生物学—分子生物学] R329.25[医药卫生—人体解剖和组织胚胎学]