泽泻法呢基焦磷酸合酶原核表达、功能验证及其免疫检测研究  

Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale(Sam.) Juzep.

作　　者：周晨 田荣 谷巍[1] 耿超[1] 吴启南[1] 徐飞[1] 巢建国[1] 刘琪[1] 王小浩 ZHOU Chen;TIAN Rong;GU Wei;GENG Chao;WU Qi-nan;XU Fei;CHAO Jian-guo;LIU Qi;WANG Xiao-hao(School of Pharmacy,Nanjing University of Chinese Medicine,Nanjing 210023,China)

机构地区：[1]南京中医药大学药学院,江苏南京210023

出　　处：《药学学报》2018年第9期1571-1577,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81673534);江苏省自然科学基金资助项目(BK20161576);江苏省中药优势学科Ⅱ期建设项目(PAPD-2014);江苏省中药资源产业化过程协同创新中心项目(ZDXM-3-24)

摘　　要：泽泻法呢基焦磷酸合酶[farnesylpyrophosphatesynthaseofAlismaorientale(Sam.)Juzep.,Ao FPPS]是泽泻原萜烷型三萜生物合成途径中的重要限速酶之一。为进一步研究Ao FPPS基因的表达及其功能,本研究将前期获得的泽泻法呢基焦磷酸合酶基因(accessionNo.HQ724508)插入到原核表达载体,构建重组表达载体p Czn1-Ao FPPS,转化BL21原核宿主菌,经诱导表达获得融合蛋白;利用Ni树脂亲和纯化技术对融合蛋白进行纯化获得高纯度的重组蛋白,并进行体外酶促反应以验证其功能,高效液相色谱结果表明Ao FPPS能够催化法呢基焦磷酸(farnesyl pyrophosphate, FPP)的合成。为进一步研究其表达规律,将该纯化后的重组蛋白免疫新西兰兔制备多克隆抗体,酶联免疫吸附测定(enzymelinkedimmunosorbentassay,ELISA)检测抗体具高效价,且Westernblot结果显示该抗体可特异性识别Ao FPPS蛋白,建立了Ao FPPS的快速免疫检测方法。本研究为揭示Ao FPPS在泽泻体内的作用机制及其基因的调控与表达奠定基础,为利用植物基因工程提高泽泻资源性活性成分含量、改善中药材品质提供科学依据。Farnesyl pyrophosphate synthase of Alisma orientale（Sam.） Juzep.（Ao FPPS） is considered as one of the important rate-limiting enzymes in the biosynthetic pathway of protostane triterpenes. In order to investigate the expression and function of Ao FPPS, the gene（accession No. HQ724508） was cloned into a bacterial expression vector p Czn1, then the combined plasmid p Czn1-Ao FPPS was transformed into Escherichia coli BL21, and a fusion protein was obtained after induction. The fusion protein was purified by Ni resin, and the function was verified through in vitro enzymatic reaction. High performance liquid chromatography（HPLC） analysis revealed that the products were able to catalyze the synthesis of farnesyl pyrophosphate（FPP）. High purity recombinant protein was used to immunize New Zealand rabbits to generate a polyclonal antibody. The titer of the antibody was determined by enzyme linked immunosorbent assay（ELISA）, and Western blot results demonstrated that the antibody could specifically recognize the Ao FPPS protein in A. orientale（Sam.） Juzep. So, the method of rapid immunoassay to detect Ao FPPS was established. This study lays the foundation for further study of the Ao FPPS gene expression, regulation and mechanism of action in A. orientale（Sam.） Juzep., and it also provides a scientific basis on improving the quality of Alismatis Rhizoma using the plant genetic engineering.

关 键 词：泽泻 法呢基焦磷酸合酶 原核表达 功能验证 免疫检测 

分 类 号：R931[医药卫生—生药学]