[Bioinformatics analysis, prokaryotic expression and biological activity of HSP70 domain-containing proteins derived from pollen of Populus deltoides].

Objective To study the bioinformatics characteristics of HSP70 domain proteins derived from pollen of Populus deltoides (P. deltoides), optimize the prokaryotic expression methods, and identify the biological activity of these proteins. Methods Physicochemical characteristics of three kinds of HSP70 domain-containing proteins were analyzed by bioinformatics software. The T/B cell epitopes of these proteins were predicted by Immune Epitope Database and Analysis Resource (IEDB). According to the amino acid sequence provided by Uniprot database, their nucleotide sequences were synthesized and cloned into pET28a(+) plasmid for prokaryotic expression. Protein expression was detected by SDS-PAGE, then the expressed products were purified by nickel column and identified by Western blotting. The protein concentration was measured by protein quantitative kit. Then the three proteins were used as antigens to prepare mouse asthma models, and the concentration of serum total IgE antibody was determined by ELISA. Results The bioinformatics analysis showed that the relative molecular mass (Mr) of B9N9W6, B9GX02 and A0A2K2AYN8 were 71 900, 94 600 and 75 200, respectively. The 13 T-cell epitopes and 14 B-cell epitopes were identified in the three proteins which had high hydrophilia and stability. SDS-PAGE analysis revealed that the genes encoding the three proteins were expressed with three specific bands of approximately Mr 72 000, 95 000 and 75 000, respectively. Western blotting showed the specific bands at the corresponding sites. ELISA showed that the IgE level in the extract group and the A0A2K2AYN8 group were higher than that in the PBS group. Compared with the A0A2K2AYN8 group, the IgE concentration in the B9N9W6 group and B9GX02 group increased significantly. Conclusion The soluble HSP70 domain-containing proteins A0A2K2AYN8, B9GX02 and B9N9W6 derived from pollen of P. deltoides can be expressed as well as purified, and have the biological activity of producing IgE antibodies.