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

Chen ZHOU; Rong TIAN; Wei GU; Chao GENG; Qi-nan WU; Fei XU; Jian-guo CHAO; Qi LIU; Xiao-hao WANG

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

VernacularTitle: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
Author: Chen ZHOU ¹ ; Rong TIAN ¹ ; Wei GU ¹ ; Chao GENG ¹ ; Qi-nan WU ¹ ; Fei XU ¹ ; Jian-guo CHAO ¹ ; Qi LIU ¹ ; Xiao-hao WANG ¹
Author Information

1. School of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
Publication Type:ORIGINAL ARTICLES
Keywords: Alisma orientale; farnesyl pyrophosphate synthase; prokaryotic expression; function verification; immunoassay
From: Acta Pharmaceutica Sinica 2018;53(9):1571-1577
CountryChina
Language:Chinese
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.