蛇足石杉内生真菌Shiraia sp.Slf14中赖氨酸脱羧酶基因的克隆、表达及功能

Molecular cloning,expression and characterization of lysine decarboxylase gene of endophytic fungus Shiraia sp.Slf14 from Huperzia serrata

【目的】阿尔茨海默症治疗药物石杉碱甲(Huperzine A,Hup A)的生物合成途径起始于赖氨酸脱羧酶(Lysine decarboxylase,LDC)。本研究克隆及表达了来源于产Hup A的植物内生真菌的LDC基因,并研究了其功能。【方法】采用RT-PCR扩增法,从一株产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14获得LDC基因,构建表达质粒p ET-22b-LDC与p ET-32a-LDC,转化感受态细胞E.coli BL21,加入IPTG至终浓度为1×10^(–3) mol/L,于24°C、200 r/min培养8 h,诱导表达LDC蛋白质;通过Ni^(2+)金属亲和层析纯化重组LDC并建立酶促反应体系,利用TLC检测了LDC催化活性。利用生物信息学软件分析了LDC的理化性质及蛋白质的空间结构。【结果】成功克隆并异源表达出重组蛋白LDC与Trx-LDC,经SDS-PAGE电泳鉴定分子量分别为24.4 k Da和42.7 k Da,与预计大小相符。TLC结果表明LDC与Trx-LDC均具有赖氨酸脱羧酶活性。【结论】本研究从产Hup A的蛇足石杉内生真菌Shiraia sp.Slf14中成功克隆到LDC基因并进行了异源表达,检测到了其催化活性,为丰富LDC分子信息及阐明内生真菌中Hup A生物合成机制提供参考数据。

[Objective] Huperzine A（Hup A） was approved as a drug for the treatment of Alzheimer＇s disease.The Hup A biosynthetic pathway was started from lysine decarboxylase（LDC）,which catalyzes lysine to cadaverine.In this study,we cloned and expressed an LDC gene from a Hup A-producing endophytic fungus,and tested LDC activities.[Methods] An endophytic fungus Shiraia sp.Slf14 from Huperzia serrata was used.LDC gene was obtained by RT-PCR,and cloned into p ET-22b（＋） and p ET-32a（＋） vectors to construct recombinant plasmids p ET-22b-LDC and p ET-32a-LDC.These two recombinant plasmids were transformed into E.coli BL21,cultured for 8 h at24 °C,200 r/min with 1×10^（–3） mol/L IPTG into medium to express the LDC proteins,respectively.LDC proteins were purified by Ni^（2＋） affinity chromatography.Catalytic activities were measured by Thin Layer Chromatography.At last,the physicochemical properties and structures of these two LDCs were obtained by bioinformatics software.[Results]LDC and Trx-LDC were expressed in E.coli BL21 successfully.SDS-PAGE analysis shows that the molecular weight of LDC and Trx-LDC were 24.4 k Da and 42.7 k Da respectively,which are consistent with bioinformatics analysis.In addition,TLC analysis reveals that both LDC and Trx-LDC had catalytic abilities.[Conclusion] This work can provide fundamental data for enriching LDC molecular information and reveal the Hup A biosynthetic pathway in endophytic fungi.