猪H1foo基因的克隆与真核表达载体的构建  

Cloning of Swine H1foo(Oocyte-specific Linker Histone H1) Gene and Construction of Eukaryotic Expression Vector pVenus-H1foo

作　　者：李伟 吴苏君[1] 杨文琳[1] 史芸安[1] 井勇[1] 赵贵民[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：《农业生物技术学报》2012年第1期85-93,共9页Journal of Agricultural Biotechnology

基　　金：陕西省2009年13115科技创新工程重大科技专项(No.2009ZDKJ-23)

摘　　要：卵特异性连接组蛋白(oocyte-specific linker histone H1,Hlfoo)是在哺乳动物卵母细胞与早期胚胎内特异表达的连接组蛋白,它在卵母细胞生长成熟、受精及胚胎发育中起关键性作用。本研究旨在克隆猪H1foo基因,并构建其真核表达载体。首先利用5'RACE和RT-PCR的方法获得猪H1foo基因的CDS区,并提交GenBank,登录号为HQ915640;然后将H1foo基因的CDS区连接到载体pMD19-T上,经酶切后定向克隆到表达载体pVenus上,从而构建pVenus-H1foo真核表达载体;用脂质体2000介导重组质粒pVenus-H1foo转染Hela细胞,荧光显微镜下观察、RT-PCR检测,确定重组质粒在Hela细胞内的表达和定位;最后通过体外转录试剂盒将H1foo-venus体外转录为mRNA,并显微注射至猪卵母细胞,荧光显微镜观察其表达和定位。序列分析表明猪Hlfoo基因CDS区全长1041bp,编码346个氨基酸,蛋白分子量为36.45kD,在核苷酸水平上与牛、人和小鼠H1foo基因的相似度分别为75.7%、67.9%和54.3%;真核表达载体pVenus-H1foo转染后,能在Hela细胞中表达并可以准确的定位在细胞核;体外转录的H1foo-venus mRNA显微注射猪卵后其融合蛋白也能准确定位于细胞核。本研究克隆了猪H1foo基因并成功构建其真核表达载体;体外转录的H1foo-venus mRNA能在猪卵中正确表达和定位,为进一步研究H1foo在猪卵母细胞成熟以及核移植过程中的作用提供了基础资料。Hlfoo（oocyte-specific linker histone H1） is a linker histone specifically expressed in oocytes and early embryos in mammalian,it plays a crucial role in oogenesis,fertilization and embryogenesis.The objectives of this study were to clone swine H1foo gene and construct a eukaryotic expression vector pVenus-H1foo.Firstly,we applied the 5＇RACE and RT-PCR to obtain the complete CDS of swine H1foo gene,the sequence information was submitted to GenBanK（GenBank accession No.：HQ915640）.The CDS of swine H1foo gene was linked into pMD19-T and confirmed by digestion of restriction enzyme,and then the H1foo CDS was cloned into pVenus to construct a eukaryotic expression vector pVenus-H1foo.After pVenus-H1foo transfected into Hela cells mediated by Lipofectamine 2000,it was efficiently expressed in hela cells and identified by observation of fluorescence microscopy and RT-PCR.In addition,after transcripted in vitro,the mRNA of H1foo-venus was microinjected into swine oocytes,the expression and location was identified under fluorescence microscopy.The result of sequence analysis showed that the CDS region of the swine H1foo gene included 1 041 nucleotides,encoding for 346 amino acids（Mol.wt.：36.45kD）,and the nucleotide similarity of H1foo between swine and bovine,human,mice was 75.7%,67.9% and 54.3%,respectively.After transfection and microinjection,the H1foo was expressed and localized accurately both in Hela cell nucleus and swine oocyte nucleus.We had cloned swine H1foo gene and constructed the expression vector pVenus-H1fooin;The mRNA of H1foo-venus transcripted in vitro was expressed and localized accurately in swine oocytes,which greatly facilitates the further research of H1foo in oocytes maturation and nuclear transplantation.

关 键 词：H1foo基因 5'RACE 体外转录 显微注射 猪卵母细胞 

分 类 号：Q782[生物学—分子生物学]