微量牛卵中H1foo CDS序列的克隆及其mRNA向卵内显微注射

摘要

This study was performed for the optimization of gene cloning from trace bovine oocytes and the construction of an eukaryotic expression vector of bovine H1foo gene. The efficiency of gene cloning by improved RT-PCR was tested. The results showed that the percentage of success-ful amplification from single oocyte was above 80% (10/12),and 100% in both 3 and 5 occytes. The complete coding sequences of H1foo was also cloned from 5 bovine oocytes,which was total-ly identical with previous reports. H1foo CDS was inserted into pMD19-T vector and confirmed by restriction enzyme digestion and sequencing, then the eukaryotic expression vector pVenus-H1foo was constructed. The recombinant plasmid pVenus-H1foo was transfected into Hela cells. H1foo was efficiently expressed in Hela cells identified by Fluorescence microscopy observation, RT-PCR and Western Blotting. H1foo was localized accurately on chromosomes of the oocyte and polar body after H1foo mRNA transcripted in vitro was micro-injected into bovine oocytes. The bovine H1foo gene eukaryotic expression vector was successfully constructed,and our study will lay a good foundation for further studying the functions of H1foo, especially its role on oo-cyte maturation and early embryo development.%本研究旨在优化从微量卵中克降基因的条件,并构建牛H1foo基因真核表达载体.首先将微量(1～5个)卵母细胞裂解后,用改进的RT-PCR方法扩增内参β-actin基因以检测该方法扩增基因的效率,结果表明单卵扩增成功率为80%以上(10/12),3个、5个卵扩增成功率均达100%.利用该体系从微量(5个)牛卵中克隆得到H1fooCDS全长序列,测序结果显示其与GenBank上公布序列同源性为100%.将牛H1foo CDS连接至pMD19-T载体上,经酶切、测序鉴定正确后定向亚克隆到真核表达载体pVenus上,鉴定正确后命名为pVenus-H1foo.利用脂质体2000将pVenus-H1foo转染Hela细胞,24 h后通过荧光显微镜观察、RT-PCR、Western Blotting分别检测表明其能够正确表达.将H1foo体外转录为mRNA后直接注射卵母细胞,其表达产物能准确定位于卵母细胞及极体的染色体上.结果,成功构建了牛H1foo真核表达载体pVenus-H1foo,为研究该基因在卵母细胞成熟及早期胚胎发育过程中的作用奠定了基础.