水牛Tle6基因表达模式分析及其多克隆抗体制备

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

【目的】明确水牛Tle6基因表达组织特异性及其在卵母细胞和早期胚胎中的表达模式,并通过构建原核表达载体诱导表达融合蛋白及免疫小鼠制备TLE6多克隆抗体,为进一步揭示Tle6基因在水牛生殖发育中的作用机制提供理论依据。【方法】采用RT-PCR扩增水牛Tle6基因编码区(CDS)序列,经生物信息学分析后,分别以半定量PCR和实时荧光定量PCR检测分析水牛Tle6基因表达组织特异性及其在卵母细胞和早期胚胎中的表达模式。构建重组原核表达载体,以IPTG诱导表达的融合蛋白免疫小鼠制备TLE6多克隆抗体,再利用TLE6多克隆抗体检验TLE6蛋白在水牛不同组织及卵母细胞和早期胚胎中的表达情况。【结果】水牛Tle6基因CDS序列全长1731 bp,编码576个氨基酸残基,其编码蛋白分子量为64.09 kD,理论等电点(pI)为5.69,属于亲水性蛋白。Tle6基因仅在水牛的卵母细胞中特异性表达。重组原核表达载体pET-32a-Tle6转化BL21(DE3)感受态细胞,经IPTG诱导6 h,融合蛋白TLE6的表达量最高,且以可溶性蛋白和包涵体2种形式进行表达;以纯化的融合蛋白TLE6免疫小鼠成功制备获得TLE6多克隆抗体,其抗体效价为1∶64000,能与融合蛋白TLE6及水牛卵母细胞发生特异性反应,即具有很强的特异性。【结论】Tle6基因仅在水牛卵母细胞中特异性表达,而在其他组织中未见表达。不同于其他MEGs的表达模式,Tle6基因在水牛卵母细胞及胚胎早期发育过程中呈特异性持续表达,可能在水牛卵母细胞成熟及附植前的胚胎发育过程中发挥重要作用。

【Objective】Characterization of the Tle6 gene expression profile and its expression in different tissues,oo-cytes and preimplantation embryos in buffalo were studied.TLE6 polyclonal antibody was prepared by constructing a pro-karyotic 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.【Methods】The sequence of Tle6 gene coding region(CDS)of buffalo was amplified by RT-PCR.After bioinformatics analysis,a prokaryo tic 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 recom-binant pET-32a-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 was 1∶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,indica-ting that may be function in oocyte maturation and preimplantation embryo development in buffalo.