水牛波形蛋白基因克隆及其表达分析

[目的]克隆水牛波形蛋白(VIM)基因,并明确VIM基因在水牛成熟卵母细胞(MII期)和早期胚胎(2-细胞阶段)中的表达情况,为研究VIM在卵母细胞成熟及附植前胚胎发育中的作用机理打下基础.[方法]根据GenBank上已公布的水牛VIM基因mRNA序列设计引物,采用RT-PCR克隆水牛VIM基因,利用在线软件程序对克隆获得的水牛VIM基因进行生物信息学分析,同时以细胞免疫荧光试验检测VIM在水牛成熟卵母细胞(MII期)及早期胚胎(2-细胞阶段)中的表达情况.[结果]水牛VIM基因长度为1469 bp,其编码区长度1401 bp,编码466个氨基酸.水牛VIM基因序列与人类、黑猩猩、食蟹猴、家犬、马和牛的VIM基因序列具有高度的同源性,分别为93%、93%、93%、94%、94%和99%;基于VIM基因序列构建的系统发育进化树也显示水牛与牛的亲缘关系最近.水牛VIM的分子量为53.73 kD,理论等电点(pI)5.05,主要在线粒体中表达,稳定性差(不稳定系数53.65),具有较强的亲水性,无跨膜结构,不属于分泌型蛋白,其蛋白结构以α-螺旋为主.VIM在水牛成熟卵母细胞(MII期)及早期胚胎(2-细胞阶段)中均有表达,且在成熟卵母细胞中的表达量明显高于早期胚胎.[结论]水牛VIM基因编码序列具有较高的保守性,且在水牛成熟卵母细胞(MII期)中的表达量明显高于早期胚胎(2-细胞阶段).

Cloning and expression analysis of buffalo vimentin gene

[Objective]Buffalo vimentin(VIM)gene was cloned and its expression was identified in buffalo mature oocyte(MII stage)and early embryo(2-cell stage)to lay a foundation for studying the mechanism of VIM in oocyte matu-ration and pre-implantation embryo development.[Method]Primer was designed according to the published mRNA se-quence of buffalo gene VIM in GenBank.The buffalo gene VIM was cloned by RT-PCR and the bioinformatics analysis was performed by using various online software programs.Meanwhile,the immunofluorescence assay was used to detect expression of VIM in buffalo mature oocytes(MII stage)and early embryo(2-cell stage).[Result]The length of the buffa-lo gene VIM was 1469 bp,the length of the coding region was 1401 bp,and 466 amino acids were encoded.The sequence of buffalo gene VIM had high homology with the VIM gene sequence of Homo sapiens(93%),Pan troglodytes(93%), Macaca fascicularis(93%),Canis lupus familiaris(94%),Equus caballus(94%)and Bos taurus(99%).The constructed phylogenetic tree based on VIM gene sequence showed that the relationship between the buffalo and the B.taurus was the closest.Buffalo VIM had a molecular weight of 53.73 kD and a theoretical isoelectric point(pI)of 5.05,which was main-ly expressed in mitochondria,with poor stability(instability coefficient of 53.65).It had strong hydrophilicity,no trans-membrane structure,and was not a secretory protein.Its protein structure was mainly composed of α-helix.VIM was ex-pressed in buffalo mature oocytes(MII stage)and early embryos(2-cell stage),and the expression level in mature oocytes was obviously higher than that in early embryos.[Conclusion]The coding sequence of buffalo gene VIM is highly con-served,and the expression level in buffalo mature oocyte(MII stage)is obviously higher than that in early embryo(2-cell stage).