绵羊GPx5基因克隆、生物信息学分析及表达特性的研究

Clone,Bioinformatics Analysis and Expression Characteristics of Ovine GPx5

本研究旨在克隆绵羊硒非依赖性谷胱甘肽过氧化酶5(GPx5)基因,预测蛋白结构与功能,并研究其在睾丸和附睾头中的表达定位特点。采用5′RACE和RT-PCR克隆GPx5基因,用生物信息学分析预测GPx5结构、特性和功能,采用免疫组化和Western blotting方法进行表达定位研究。结果显示,绵羊GPx5的开放阅读框(ORF)660bp(GenBank:JQ 320282),GPx5编码219aa,分子量约25ku的碱性蛋白,第1~21个aa为潜在的信号肽,其三级结构域为αβ型,有11个可能磷酸化位点,有7种保守的特异性蛋白质激酶的结合位点;亚细胞定位预测显示GPx5存在于细胞外;其38~151aa属于谷胱甘肽过氧化酶家族保守结构域,其36~215aa类硫氧还蛋白超家族保守结构域。绵羊GPx5在附睾头的假复层柱状上皮细胞大量表达,附睾头上皮细胞和睾丸间质细胞少量表达,附睾头液和精浆中均有GPx5蛋白。绵羊GPx5是分泌型,具有谷胱甘肽过氧化酶活性和类硫氧还蛋白作用。GPx5可能是某信号转导通路的信号分子,但需进一步验证。

The aim of this study was to clone ovine GPx5(Selenium-independent glutathione peroxidase 5),and predict the structure and function of GPx5 protein,and investigate its expression and location characteristics in ovine testis and epididymis caput.The ovine GPx5 was cloned by 5′RACE and RT-PCR.The structure,characteristics and function of ovine GPx5 protein were analyzed by bioinformatics methods.The expression characteristics were detected by immunohistochemistry assays in ovine testis and epididymis caput,as well as by Western blotting assays in seminal plasma.The results showed GPx5gene(GenBank accession No.:JQ 320282)contained an660 bp open reading frame,which coded 219 aa with molecular weight of 25 ku.Its potential signal peptide was 1-21 aa.The domain structure of tertiary structure was alpha/beta structure.There were 11 potentail phosphorylation sites predicted and 7conserved and specific phosphokinase binding sites.The prediction results of InterProScan showed 38-151 aa were the conserved protein domain of GSHPx family and 36-121 aa was the conserved protein domain of thioredoxin-like su-perfamily.GPx5 protein was essentially presented in pseudostratified columnar epithelium,and poorly presented in epithelium of the ovine epididymis and Leydig cell of ovine testis,and also presented in fluid of epididymis caput and seminal plasma in rams.Ovine GPx5 protein was secreted,which had a GPx activity and function of thioredoxin-like.GPx5 could be as a mediator of signaling pathway and need be tested by further research.