药学学报, 2018, 53(9): 1571-1577
引用本文:
周晨, 田荣, 谷巍, 耿超, 吴启南, 徐飞, 巢建国, 刘琪, 王小浩. 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[J]. 药学学报, 2018, 53(9): 1571-1577.
ZHOU Chen, TIAN Rong, GU Wei, GENG Chao, WU Qi-nan, XU Fei, CHAO Jian-guo, LIU Qi, WANG Xiao-hao. Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep.[J]. Acta Pharmaceutica Sinica, 2018, 53(9): 1571-1577.

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
周晨, 田荣, 谷巍, 耿超, 吴启南, 徐飞, 巢建国, 刘琪, 王小浩
南京中医药大学药学院, 江苏 南京 210023
摘要:
泽泻法呢基焦磷酸合酶[farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep.,AoFPPS]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AoFPPS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(accession No.HQ724508)插入到原核表达载体,构建重组表达载体pCzn1-AoFPPS,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AoFPPS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(enzyme linked immunosorbent assay,ELISA)检测抗体具高效价,且Western blot结果显示该抗体可特异性识别AoFPPS蛋白,建立了AoFPPS的快速免疫检测方法。本研究为揭示AoFPPS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。
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     
Prokaryotic expression, function verification and immunoassay activity of farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep.
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
Abstract:
Farnesyl pyrophosphate synthase of Alisma orientale (Sam.) Juzep. (AoFPPS) 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 AoFPPS, the gene (accession No. HQ724508) was cloned into a bacterial expression vector pCzn1, then the combined plasmid pCzn1-AoFPPS 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 AoFPPS protein in A. orientale (Sam.) Juzep. So,the method of rapid immunoassay to detect AoFPPS was established. This study lays the foundation for further study of the AoFPPS 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.
Key words:    Alisma orientale    farnesyl pyrophosphate synthase    prokaryotic expression    function verification    immunoassay   
收稿日期: 2018-05-02
DOI: 10.16438/j.0513-4870.2018-0401
基金项目: 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).
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相关文献:
1.谷 巍 吴启南 巢建国 席蓓莉 李 琳 申修源.建泽泻法呢基焦磷酸合酶分子克隆、分布表达及生物信息学研究[J]. 药学学报, 2011,46(5): 605-612