修饰GLP2分泌性表达载体的构建及促增殖活性测定的实验研究  

Construction of secretary expression vector of mutation chimeric GLP2 and detection of its proliferation-promoting activity

作　　者：谭虎[1] 杨天德[2] 粟永萍[1] 艾国平[1] 黄岚[3] 陶军[2] 刘晓宏[1] 楼淑芬[1] 

机构地区：[1]第三军医大学军事预防医学院防原医学教研室,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 [2]第三军医大学新桥医院麻醉科,重庆400037 [3]第三军医大学新桥医院全军心血管病研究所,重庆400037

出　　处：《第三军医大学学报》2007年第17期1650-1653,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金重点项目(30230360);国家自然科学基金面上项目(30470527);创伤;烧伤与复合伤国家重点实验室开放课题(2003)~~

摘　　要：目的对胰高血糖素样肽-2(glucagon-like peptid-2,GLP-2)进行基因修饰,观察其促细胞增殖活性。方法通过RT-PCR方法获得含GLP2编码序列的高血糖素原cDNA片段;通过PCR重叠延伸技术得到GLP2信号肽及成熟肽的嵌合分子,并将编码GLP2成熟肽N-末端第二位丙氨酸的序列(GCT)突变为编码甘氨酸的序列(GGT);将其插入真核表达质粒pVITRO3,转染至肠上皮细胞(Caco-2),观察其对细胞增殖活性的影响。结果克隆的修饰GLP2基因与国外报道的核苷酸序列及其编码的氨基酸序列完全一致,并成功拼接GLP2的信号肽及成熟肽;且加入pVITRO3-GLP2-Caco-2培养上清的细胞生长明显快于对照组。结论PCR重叠延伸技术适用于分泌性载体构建。构建的修饰GLP2表达系统所表达的蛋白具有促进肠上皮细胞增殖的作用。Objective To construct the secretary expression vector of mutation chimeric GLP2 by clone technology and detect its effect on cell proliferation. Methods The proglucagon cDNA was generated by the reverse transcription. The PCR product of signal and mature peptides of GLF2 was linked by SOE （splicing by overlap extension） and the gene, which coded the second amino acid in the N-terminal of GLP2, was mutated from GCT to GGT by PCR. Then the chimeric GLF2 cDNA was cloned into pVITRO3 vector and the recombinant plasmid was transfected to Caco-2. The cell proliferation was detected by MTT. Results The sequences of signal peptide and mature peptide of GLP2 were connected successfully and the mature peptide sequence was mutated. MTT and 3 H-TdR showed that rGLF2 could accelerate the proliferation of Caco-2 cell significantly. Conclusion SOE is appropriate in establishing secretion type of expression vector and the secretary expression vector of mutation chimeric GLP2 has accelerating effect on cell proliferation.

关 键 词：胰高血糖素样肽-2 基因克隆 PCR重叠延伸技术 肠上皮 细胞增殖 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学] R394-33[医药卫生—基础医学]