不育相关泛素特异性蛋白酶USP26的分子克隆及活性分析

Molecular cloning and activity analysis of infertility-associated gene ubiquitin-specific protease 26

目的对克隆泛素特异性蛋白酶26(ubiquitin-specific protease 26,usp26)基因进行表达和活性测定。方法采用聚合酶链式反应(polymerase chain reaction,PCR),从小鼠全血中扩增获得usp26基因并测定序列。将基因片段与p GEX-6p-1拼接,应用异丙基-β-d-硫代半乳糖苷(isopropyl-β-d-thiogalactoside,IPTG)使蛋白表达并利用Ub-Met-β-gal鉴定活性。用多重比对方法分析人类、小鼠及褐家鼠usp26基因编码氨基酸序列的差异。结果本实验克隆出usp26基因。构建出p GEX-usp26质粒,该质粒表达出GST-USP26结合蛋白并测得其活性。三个种属含有相同的赖氨酸结构域(Cys box)与组氨酸结构域(His box)特征部位氨基酸。结论 usp26基因克隆并表达成功,完成活性测定,为进一步探索usp26在男性不育机制方面的作用提供了一种活性研究方法。

Objective To clone the ubiquitin-specific protease 26( usp26),express and detect its activity.Methods usp26 was amplified from rat genome by PCR for sequence analysis.We spliced together the amplified fragment and p GEX-6p-1 and then expressed protein under the isopropyl-β-d-thiogalactoside( IPTG) induction.Furthermore we detected the enzyme activity using Ub-Met-β-gal.The amino acid sequences of usp26 between Homo sapiens and Mus musculus as well as Rattus norvegicus were multiplely compared.Results This study had cloned usp26; built p GEX-usp26 plasmid;expressed GST and USP26 protein and tested activity of enzyme.Characteristic parts of conserved amino acids( Cys and His boxes) in three species were the same.Conclusions We cloned usp26 gene successfully,completed the enzyme activity determination and provided a research method to further explore the role of usp26 in the mechanism of infertility.