Bioinformatics analysis and prokaryotic expression of apical membrane antigen protein AMA1 of Isospora suis

Objective  To predict and analyze the biological characteristics, structure and function of the apical membrane antigen 1 (AMA1) in Isospora suis, as well as construct the prokaryotic expression vector of AMA1 gene to express and purify AMA1 protein.

Method The AMA1 gene sequence of I. suis was obtained from NCBI database, and the protein encoded by AMA1 gene was analyzed using relevant bioinformatics prediction tools. The recombinant prokaryotic expression vector pET23a-AMA1 was constructed and transferred into the expression strain BL21 (DE3) of Escherichia coli. The induction time, temperature and IPTG concentration were optimized to determine the optimal induction expression conditions. The recombinant AMA1 protein was purified by nickel column affinity chromatography and identified by SDS-PAGE and Western blot.

Result Bioinformatics prediction showed that AMA1 protein was composed of 317 amino acids, and its molecular formula was C₁₅₁₂H₂₃₁₀N₃₉₄O₄₉₀S₁₄, which was an unstable hydrophilic protein. The secondary structure prediction of the AMA1 protein yielded a profile consisting of 17.74% α helix, 30.65% β folding, 30.65% rotation, and 20.96% random coil. It was a transmembrane protein without signal peptide and had five B cell epitopes. At the same time, pET23a-AMA1 recombinant expression plasmid was successfully constructed, and the optimal induction expression condition was 0.2 mmol/L IPTG inducing for 12 h at 28 ℃, which mainly existed in the form of soluble protein. The relative molecular mass of recombinant protein was about 25 800, and the mass concentration of purified protein was 0.25 mg/mL.

Conclusion The results of this study elucidate the structural characteristics of AMA1 protein of I. suis and the recombinant protein is obtained through prokaryotic induction expression, which lays a foundation for the establishment of immunological diagnosis of I. suis and provides a new candidate gene for the development of subsequent vaccines.