冷诱导rna结合蛋白cirp过表达载体的构建-应用与环境生物学报.docVIP

冷诱导RNA结合蛋白（CIRP）过表达载体的构建及鉴定* 李静辉1 孟 宇1 李玉恒1 李昌盛1 杨焕民1 李士泽1, 2** 1.黑龙江八一农垦大学动物科技学院 ；2. 黑龙江八一农垦大学食品学院 大庆 163319 摘 要 构建携带冷诱导RNA结合蛋白(Cold inducible RNA-binding protein，CIRP)的过表达载体，为进一步研究 CIRP 的作用机制奠定良好的实验基础。首先从4 ℃条件下冷处理8 h的SD大鼠睾丸组织中获得 CIRP 基因的 cDNA 序列，然后扩增 CIRP 基因的编码区，将其克隆到真核表达载体pLenti6/V5中，酶切鉴定并测序。将克隆成功的质粒转染HEK293T细胞，用western blot 检测CIRP 的表达水平。本试验成功扩增了 CIRP 编码区，并将其克隆至载体 pLenti6/V5 中；当将CIRP 过表达载体转染 HEK293T 细胞后，western blot 检测结果显示 CIRP 蛋白表达水平明显增加(P0.01)。本研究成功构建了CIRP过表达载体，为今后的研究提供了基础工具。 图4表1参21 关键词 冷诱导RNA结合蛋白；过表达；质粒；冷应激 CLC Q494 : Q78 Construction and Identification of Over-expressing Vector of Cold inducible RNA-binding Protein (CIRP)* LI Jinghui1, MENG Yu1, LI Yuheng1, LI Changsheng1, YANG Huanmin1＆LI Shize1,2** College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China 2.College of food, Heilongjiang Bayi Agricultural University, Daqing 163319, China Abstract Objective: To provide a basis tool for further research of CIRP function, a vector for the over-expression of Cold inducible RNA-binding Protein (CIRP) was constructed in this study. After obtained CIRP cDNA sequencing of testicular tissue from SD rats with cold treated at the temperature of 4 ℃, the coding region of CIRP gene was amplified and then cloned into the eukaryotic expression vector pLenti6/V5, followed by endonuclease digestion and sequencing. The successful cloned plasmid was transfected into HEK293T cells, and CIRP expression was determined by Western blot. The coding region of CIRP gene was successfully amplified, and cloned into the pLenti6/V5. After transfected with CIRP over-expression vectors，expression of CIRP protein was up-regulated in HEK293T cells (P0.01). This study indicated that over-expression vector of CIRP was successfully constructed and determined, provided a basis tool for further research of CIRP. Key words CIRP; over-expression; plasmid;cold stress 冷诱导RNA结合蛋白(Cold inducible RNA-binding protein，CIRP)是在哺