Prokaryotic Expression of Hcp Protein of Aeromonas hydrophila,Preparation of Polyclonal Antibody and Bioinformatics Analysis
[Objective]In order to obtain a polyclonal antibody to the Hcp protein of Aeromonas hydrophila(AH)and its basic biological properties.[Method]The present study utilized RT-PCR to amplify the Hcp gene,constructed a recombinant expression plasmid and then trans-formed the BL21 receptor cells,which were induced to express the Hcp recombinant protein through IPTG(Isopropyl β-D-Thiogalactoside),purified and immunized rabbits,and then prepared a polyclonal antibody against the protein.Hcp recombinant protein was obtained by IPTG(Isopropyl β-D-Thiogalactoside)induced expression,purified and immunized rabbits,and polyclonal antibody against the protein was pre-pared,and the potency of the antibody was determined,identified by Western blotting,and its basic physicochemical properties,conserved structural domains,and phosphorylation sites were investigated using bioinformatics online tools,secondary and tertiary structure prediction.[Result]The results showed that the highest expression of the protein was obtained at the final concentration of IPTG of 0.6 mmol/L at 37℃for 6 h,and the protein mainly existed in the form of soluble protein;the results of Western blotting showed that the supernatant and precipitate of the culture of AH were able to specifically bind to the prepared rabbit antibody with good reactivity,and the potency of the prepared poly-clonal antibody reached 1.024×106.Bioinformatics analysis showed that the molecular formula of Aeromonas hydrophila Hcp protein was C846H1304N224O259S8,encoding a total of 172 amino acids,with a relative molecular mass of 19 013.49 u,an isoelectric point(pl)of 5.24,which is a stabilized protein;the secondary structure of the protein was 51.74％of the irregularly coiled,25.00％of the elongated chain,and 25.00％of the alpha-chain.25.00％,α-helix accounted for 19.19％,and β-turns accounted for 4.07％.[Conclusion]In this study,a poly-clonal antibody to Hcp protein was successfully prepared,which can provide technical support for the further study of Type Ⅵ secretion system(T6SS)of AH.
Aeromonas hydrophilaHcp geneProkaryotic expressionPreparation of polyclonal antibodyBioinformatics
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