人卵泡抑素基因真核表达载体构建和体外表达

Construction and in vitro expression of eukaryotic expression plasmid with human FS gene

目的:体外构建携带人卵泡抑素基因(FS)cDNA的真核表达载体,并观察其在幼年叙利亚地鼠肾细胞(BHK21)中的表达。方法:从新鲜人卵泡抽提液细胞中提取RNA,应用RT-PCR方法扩增人FScDNA序列,克隆到pMD-18T载体中,构成pMD-FS重组克隆质粒。经测序鉴定后,双酶切得到人FS基因cDNA序列,插入到真核表达载体pEGFP-N1中,构成pEGFP-FS重组表达质粒。pEGFP-FS重组表达质粒经酶切和PCR鉴定,通过脂质体2000介导瞬时转染BHK21细胞,并检测绿色荧光蛋白(GFP)的荧光表达。结果:成功构建pMD-FS重组克隆质粒,FScDNA序列测序结果与GenBank中公布的序列(Genbank NM_013409.1)同源性为100%;成功构建pEGFP-FS重组表达质粒,将其转染BHK21细胞后,观察到绿色荧光蛋白的表达。结论:本实验成功构建了人FS的pMD-FS重组克隆质粒和pEGFP-FS重组表达质粒,为进一步研究FS对哺乳动物卵泡发育的影响奠定了基础。

Objective：To construct a recombinant eukaryotic expression plasmid which contains cDNA of human follistatin（FS）and explore its expression in baby hamster syrian kidney（BHK21）cells.Methods：The total RNA was isolated from human fresh follicular fluid cells.The FS complementary DNA（cDNA）was amplified by reversal transcription polymerase chain reaction（RT-PCR）and cloned into pMD-18T vector to constructed pMD-FS recombinant cloning plasmid.After the sequence of pMD-FS was analyzed,FS cDNA was cut out by double restrictive digestion,then inserted into eukaryotic expression plasmid pEGFP-N1 to construct recombinant expression plasmid pEGFP-FS.After verified by digestion and sequence analysis,pEGFP-FS was transiently transfected into BHK21 cells by lipofectamine 2000.Then enhanced green fluorescent protein（EGFP）was detected to verify the expression of FS.Results：The pMD-FS recombinant cloning plasmid was constructed successfully,and the sequencing result of FS cDNA were 100% identical with that reported in GeneBank（NM_013409.1）.The pEGFP-FS recombinant expression plasmid was constructed successfully,and the expression of EGFP was detected after pEGFP-FS transiently transfected into BHK21 cells.Conclusion：Recombinant cloning plasmid pMD-FS and recombinant expression plasmid pEGFP-FS were constructed successfully,which lays a foundation to further study on the bio-function of FS gene to the development of mammalian ovary.