p21基因在牛卵成熟中的表达检测及其真核表达载体构建  

Detection for p21 Gene Expression in Bovine Oocytes Maturation and Construction of Eukaryotic Expression Vector pVenus-P21

作　　者：赵贵民[1] 杨文琳[1] 吴苏君[1] 云彦[1] 雷安民[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《畜牧兽医学报》2011年第8期1071-1080,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：2009-2011年西北农林科技大学青年科学基金项目(Z109021004)

摘　　要：本研究旨在检测p21基因在牛卵成熟过程中的表达,并构建其真核表达载体。首先利用RT-PCR和免疫荧光染色对不同成熟阶段牛卵内p21的mRNA和蛋白表达进行检测。然后从牛成纤维细胞中克隆了p21基因并构建pVenus-P21真核表达载体。经脂质体2000介导重组质粒pVenus-P21转染Hela细胞,通过荧光显微镜观察、RT-PCR检测,确认重组质粒在Hela细胞内的表达及定位。最后应用体外转录试剂盒将p21-venus体外转录为cRNA,并注射牛卵母细胞,荧光显微镜下观察其表达及定位。结果显示,在牛卵体外成熟过程中,各时期均存在p21基因mRNA及蛋白的表达。构建的真核表达载体pVenus-P21能够在Hela细胞中正确表达及定位。p21-venus cRNA显微注射牛卵后其融合蛋白也能实现准确定位,为研究P21在卵母细胞成熟以及胚胎发育过程中的作用奠定了基础。The objectives of this study were to determine expression of p21 gene during IVM of bovine oocytes,and construct a eukaryotic expression vector pVenus-P21.Firstly,the RT-PCR and immunohistochemical staining were applied to check the expression of p21 mRNA and protein at different maturation stages of bovine oocytes.Then the p21 gene was cloned from bovine fibroblasts by RT-PCR,and a eukaryotic expression vector pVenus-P21 was constructed.After pVenus-P21 mediated by Lipofectamine 2000,it was transfected into Hela cells,it was efficiently expressed,which was identified by Fluorescence microscopy observation and RT-PCR.In addiation,the cRNA of p21-venus transcripted in vitro was microinjected into bovine oocytes,and the expression and location was observed under fluorescence microscopy.In conclusion,these data demonstrate that p21 mRNA and protein are expressed at defferent stages of bovine oocytes.Besides,the recombinant vector pVenus-P21 is successfully constructed.After transfection or microinjection,the fusion protein could be expressed efficiently and localized accurately both in Hela cells and bovine oocytes.This greatly facilitates the further study of P21,especially its role on oocytes maturation and early embryo development.

关 键 词：牛卵母细胞 P21基因 体外转录 显微注射 

分 类 号：S823[农业科学—畜牧学] Q344.13[农业科学—畜牧兽医]