Bioinformatical Analysis, Expression,Purification And Detection Of Enzyme Activity Of Echinococcus Granulosus Fructose-bisphosphate Aldolase

Objective:The bioinformatic analysis and characterization of Echinococcus fructose bisphosphate aldolase (FBPA) structurally and functionally. And its clone, expression, purification and accessment of bioactivity.Methods:Taking advantage of a variety of bioinformatics softwares to analyze the topological, biological and immunlogical function characteristics of the Echinococcus granulosus FBPA. The EcoRI and HandⅢ were used to cut object fragment of FBPA. And then FBPA was subcloned into the expression vector pET30a, which was subsequently screened and sequenced. The correct recombinant plasmid was transformed into BL21(DE3) competent bacteria and expressed in the presence of isopropyl-beta-thiogalactoside (IPTG). The recombinant protein was purified using Ni-NTA column, and then an enzyme reaction system (nicotinamide adenine dinucleotide) was created throuth reduction of NADH to determine the enzyme catalytic activity of the recombinant protein EgFBPA.Results:EgFBPA cDNA, encoding a336amino acid protein, is composed of1092base pairs(bp). The result of software analysis showed that its isoelectric point (PI) was8.34and a molecular weight was39803.5Da. The analysis of subcellular localization showed that the protein was a stable cytoplasmic protein without signal peptide. The results of domain and conserved domain prediction found the activative site FBPAIVYLEGTLLKPN (222aa-232aa) and TIM(β/α) barrel-like structure (6aa-346aa). The prediction results idicated taht the secondary structure were composed of alpha-helix and a cyclic structure, with10kinds of function sites, but no transmembrane region. The1ADO A as a template was used to construct the tertiary structure of FBPA. Successful subclone of pEY30a (+)-EgFBPA was continued by double enzyme digestion and sequencing. The recombinant protein, which was soluble, was successfully and highly expressed. We got high purity of the FBPA by using Ni-NTA column. The protein concentration of0.50±0.02mg/ml was determined by using Bradford method.Conclusions:The homology between EgFBPA and homo FBPA is69%. EgFBPA may be potential drug targets. Recombinant plasmid pET30a (+)-FBPA in E. coli BL21is successfully expressed and purified, which probably laid a foundation for further study for biological function, antibody and drug targets.