Expression pattern of Tle6 gene in buffalo and preparation of polyclonal antibody

【Objective】Characterization of the Tle6 gene expression profile and its expression in different tissues, oocytes and preimplantation embryos in buffalo were studied. TLE6 polyclonal antibody was prepared by constructing a prokaryotic expression vector with induced fusion protein expression and immuned mice. This study laid the foundation for studying the function and regulation mechanism of Tle6 gene in buffalo reproduction development.MethodsThe sequence of Tle6 gene coding region(CDS) of buffalo was amplified by RT-PCR. After bioinformatics analysis, a prokaryotic expression vector was constructed. The mouse were immunized by IPTG induced TLE6 recombinant protein for preparing the polyclonal antibody. The expression profiles of TLE6 protein in different tissues, oocytes and preimplantation embryos of buffalo was examined using TLE6 polyclonal antibody.【Result】The CDS sequence of buffalo Tle6 gene consisted of 1731 bp, encoding 576 amino acids with the molecular weight of 64.09 kD and isoelectric point(pI) of 5.69. The TLE6 protein belonged to a hydrophilic protein. The Tle6 gene was specificly expressed in oocytes of buffalo. The recombinant pET-32 a-Tle6 prokaryotic expression vector was transfer to BL21(DE3) competent cell. TLE6 protein had highest expression by 6 h IPTG induced. TLE6 protein was expressed in two forms:soluble protein and inclusion body. The TLE6 polyclonal antibody was prepared successfully by purified fusion protein TLE6 immunity mouse. The antibody titer was1:64000. It had specific reactions with the fusion protein TLE6 and buffalo oocytes, with strong specificity.【Conclusion】The Tle6 gene is specifically expressed only in buffalo oocytes and is not expressed in other tissues. Different from other MEGs expression pattern, the buffalo Tle6 gene continuously expresses in oocytes and preimplantation embyros, indicating that may be function in oocyte maturation and preimplantation embryo development in buffalo.