Cloning and expression analysis of buffalo vimentin gene

ObjectiveBuffalo 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.MethodPrimer 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).ResultThe 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.ConclusionThe 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).