人Prx6的异源表达、酶学性质及结构分析  

Heterologous Expression,Enzymatic Properties and Structure Analysis of Human Peroxiredoxin 6

作　　者：刘馨竹 林睿 安丽萍[1] 杜培革[1] 郭笑[1] LIU Xinzhu;LIN Rui;AN Liping;DU Peige;GUO Xiao(Pharmacy College of Beihua University,Jilin 132013,China)

机构地区：[1]北华大学药学院,吉林吉林132013

出　　处：《北华大学学报（自然科学版）》2022年第1期48-52,共5页Journal of Beihua University（Natural Science）

基　　金：国家自然科学基金青年基金项目(31900918);吉林省卫生健康青年科技骨干培养计划项目(2019Q020).

摘　　要：目的构建大肠杆菌中人过氧化物氧还蛋白6(human peroxiredoxin 6,hPrx6)基因表达载体,并对其进行纯化,分析其酶学性质及结构.方法利用PCR技术调取hPrx6基因片段,与pColdI载体连接,构建重组质粒pColdI-hPrx6,并将其转化到大肠杆菌BL21(DE3)中进行表达;经SDS-PAGE检测和Western blot鉴定,表达产物通过Ni-NTA亲和层析进行纯化,并对其酶学性质进行研究;对所表达的hPrx6融合蛋白利用在线软件进行结构分析.结果重组质粒经PCR和酶切法鉴定构建成功;经Ni-NTA亲和层系纯化后得到的融合蛋白纯度可达95%;对其酶学性质的研究表明:其酶活力为(240±18)U/μmol,最适反应温度为30℃,pH为7;hPrx6酸性氨基酸总数多于碱性氨基酸总数,归类为不稳定蛋白.结论本研究成功在大肠杆菌中表达可溶性hPrx6重组蛋白,此结果为研究其生物学功能奠定了基础.Objective Human peroxiredoxin 6(hPrx6)was expressed in E.coli and its purification,enzymatic property analysis and structure analysis were carried out.Method The hPrx6 gene fragment was obtained by PCR and ligated with pColdⅠvector to construct the recombinant plasmid pColdⅠ-hPrx6.The recombinant pla-smid pColdⅠ-hPrx6 was transformed into E.coli BL21(DE3)for expression,which was detected by SDS-PAGE and identified by Western blot.The expressed product was purified by Ni-NTA affinity chromatography,then its enzymatic properties were studied.The structure of the expressed hPrx6 fusion protein was analyzed by online software.Results The recombinant plasmid was identified and constructed successfully by PCR and restriction endonuclease digestion.The purity of the fusion protein purified by Ni-NTA affinity layer was up to 95%.The enzymatic properties showed that the enzyme activity was 240±18 U/μmol,the optimum reaction temperature and pH were 30℃,7.The total number of acidic amino acids was more than the total number of basic amino acids,so it was classified as unstable protein.Conclusion The soluble hPrx6 recombinant protein was successfully expressed in E.coli,which laid a foundation for further study of its biological function.

关 键 词：过氧化物氧还蛋白6 可溶性表达 酶学性质 

分 类 号：Q554[生物学—生物化学]