艰难拟梭菌甲基化酶CamA蛋白的原核表达、纯化及其活性  

Prokaryotic expression,purification and activity of CamA protein with methylase activity from Clostridiodies difficile

作　　者：段慈东 陈毓 宋小军 林珊 金大智 DUAN Cidong;CHEN Yu;SONG Xiaojun;LIN Shan;JIN Dazhi(School of Laboratory Medicine and Bioengineering,Hangzhou Medical College,Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang Province,Hangzhou 310053,Zhejiang Province,China)

机构地区：[1]杭州医学院检验医学院与生物工程学院、浙江省生物标志物与体外诊断转化重点实验室,浙江杭州310053

出　　处：《中国生物制品学杂志》2023年第8期918-923,共6页Chinese Journal of Biologicals

基　　金：浙江省自然科学基金重点项目(LXZ22H300001);浙江省自然科学基金探索项目(LQ20H-280002);2021年度省部共建重大项目(WKJ-ZJ-2107);杭州医学院基本科研业务费基础科研项目(KYZD202004)。

摘　　要：目的 原核表达具有甲基化酶活性的艰难拟梭菌CamA(Clostridiodies difficile adenine methltransferase A,CamA)蛋白。方法 PCR扩增艰难拟梭菌1870标准菌株CamA蛋白的基因序列,克隆至质粒pGEX-4T-1-MBP中,转化至大肠埃希菌HB101,经1 mmol/L IPTG诱导表达重组目的蛋白,利用Amylose Resin纯化后,CamA蛋白再经烟草蚀纹病毒(tobacco etch virus,TEV)蛋白酶酶切,采用体外MTase-GloTMMethyltransferase Assay分析技术验证其甲基化活性。结果 PCR测序结果显示克隆的CamA基因序列正确,未发生碱基突变;重组表达质粒pGEX-4T-1-MBP-CamA经EcoRⅠ和BamHⅠ双酶切后显示1 731 bp的CamA基因已连接至载体上;表达的重组蛋白相对分子质量约为108 800(含1个MBP标签),经Amylose Resin纯化后,纯度达90%以上;在20μmol/L DNA和20μmol/L S-腺苷甲硫氨酸(S-adenosylmethionine,SAM)的条件下室温反应30 min,0.5μg CamA能产生浓度约为101.60 nmol/L的S-腺苷同型半胱氨酸(S-adenosyl-L-homocysteine,SAH),酶活性为(0.339±0.027)U/mg。结论 成功表达并纯化了艰难拟梭菌CamA蛋白,其具有甲基化酶活性,为后续进一步研究CamA的功能及其在艰难拟梭菌感染发生、发展及治疗中的作用奠定了基础。Objective To express CamA(Clostridiodies difficile adenine methltransferase A,CamA)protein with methylase activity in prokaryotic expression system.Methods The gene sequence of CamA protein of the standard strain of C.difficile1870 was amplified by PCR,cloned into plasmid pGEX-4T-1-MBP,transformed into E.coli HB101 and induced by 1 mmol/L IPTG to express the recombinant target protein. After purification with Amylose Resin,CamA protein was digested by tobacco etch virus(TEV)protease,and verified for its methylase activity using MTase-Glo~(TM)Methyltransferase Assay in vitro.Results PCR sequencing showed that the cloned CamA gene sequence was correct,in which no base mutation occurred. The recombinant expression plasmid pGEX-4T-1-MBP-CamA was digested by EcoRⅠand BamHⅠ,which showed that 1 731 bp of CamA gene was connected to the vector plasmid. The relative molecular mass of the recombinant protein was about 108 800(with 1 MBP tag),and the purity was over 90% after purified with Amylose Resin. Under the condition of20 μmol/L DNA and 20 μmol/L SAM at room temperature for 30 min,0. 5 μg CamA produced SAH at a concentration of about 101. 60 nmol/L and the enzyme activity was(0. 339 ± 0. 027)U/mg.Conclusion The CamA protein of C.difficile has been successfully expressed and purified with methylase activity,which lays a foundation of further study on the function of CamA and its role in the occurrence,development and treatment of C.difficile infection.

关 键 词：艰难拟梭菌 CamA蛋白 DNA甲基化酶 原核表达 纯化 酶活性 

分 类 号：R394.3[医药卫生—医学遗传学]