大鼠C-sis基因克隆及其真核表达载体的构建  

Cloning of rat C-sis gene and construction of its eukaryotic expression vector

作　　者：孙国芳[1] 丁浩[2] 

机构地区：[1]南昌大学第二附属医院心电诊断室,江西省南昌市330006 [2]南昌大学第二附属医院消化内科,江西省南昌市330006

出　　处：《中国组织工程研究》2016年第49期7418-7424,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金资助项目(81300348);江西省青年科学基金资助项目(20151BAB215006);江西省青年科学基金资助项目(20132BAB215015)~~

摘　　要：背景:原癌基因C-sis具有促进细胞增殖并抑制凋亡,进而促进组织修复的功能。假设C-sis可能在受损肝组织的修复和暴发性肝衰竭的治疗中发挥积极作用。目的:构建pc DNA3.1/C-sis真核表达载体,检测其在大鼠正常肝细胞株BRL细胞和在体大鼠肝脏细胞中的表达。方法:通过RT-PCR的方法克隆C-sis基因的选全长编码序列,构建pc DNA3.1/C-sis真核表达载体。鉴定无误后经脂质体介导转染到BRL细胞中,并通过将质粒注入尾静脉后导入大鼠肝脏。最后通过荧光定量PCR和Western Blot鉴定其在BRL细胞和在体大鼠肝脏细胞中的表达。结果与结论:(1)成功克隆了C-sis基因全长编码区;测序证明pcD NA3.1/C-sis重组真核表达载体构建成功;(2)将其转染至BRL细胞和在体大鼠肝脏,可使C-sis表达升高;(3)实验结果为后续研究C-sis基因对大鼠暴发性肝衰竭的影响提供了先决条件。BACKGROUND： C-sis proto-oncogene can promote tissue repair by inducing cell proliferation and inhibiting cell apoptosis. Therefore, C-sis may play a positive role in the repair of damaged liver tissue and the treatment of fulminant hepatic failure. OBJECTIVE： To construct pc DNA3.1/C-sis eukaryotic expression vector and detect its expression in BRL cells（the normal liver cells of rats） and rat liver cells in vivo. METHODS： The full-length coding sequence of C-sis gene was cloned through real time-PCR. pc DNA3.1/C-sis eukaryotic expression vector was constructed and sequenced, followed by transfected into BRL cells using liposome and injected into the rat liver via tail vein. Finally, its expression in BRL cells and rat liver cells in vivo was identified by fluorescence quantitative PCR and western blotting. RESULTS AND CONCLUSION：（1） The full length of encoding region of C-sis gene was successfully cloned. Sequencing proved that pcD NA3. 1/C-sis recombinant eukaryotic expression vector was constructed successfully.（2） The expression of C-sis was increased after transfected into BRL cells and rat liver.（3） These results provide basis for the subsequent study of the effect of C-sis gene on fulminant hepatic failure in rats.

关 键 词：动物 模型 基因 脂质体 转染 组织工程 实验动物 基因病毒载体相关因子模型 基因克隆 C-SIS 大鼠 真核表达载体 酶切 鼠尾静脉 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]