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

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.