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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 and express ubiquitin-specific protease 26 (usp26) gene. Methods The usp26 gene was amplified from whole mouse blood by polymerase chain reaction (PCR) and sequenced. The gene fragment was ligated to pGEX-6p-1 and the protein was expressed using isopropyl-β-d-thiogalactoside (IPTG) and identified by Ub-Met-β-gal active. The amino acid sequences of usp26 gene in human, mouse and Rattus norvegicus were analyzed by multiple alignment method. Results The experiment cloned usp26 gene. The pGEX-usp26 plasmid was constructed which expresses the GST-USP26 binding protein and measures its activity. Three species contain the same lysine domain (Cys box) and histidine domain (His box) characteristic amino acids. Conclusion The usp26 gene was cloned and expressed successfully, and the activity assay was completed. It provides an active method for further exploring the role of usp26 in male infertility mechanism.