Recombinant expression and immunological characterization of Burkholderia pseudomallei type Ⅲ secretion system protein BipD

Abstract

Objective To express recombinant Burkholderia pseudomallei (B. pseudomallei) type Ⅲ secretion system BipD protein, prepare its polyclonal antibodies and verify their immunological traits. Methods The recombinant pET-28a-BipD plasmid was generated, and the pET-28a-BipD-carried E.coli BL21 (DE3) bacteria were induced with isopropyl-β-d-thiogalactoside (IPTG) to express recombinant BipD (rBipD) protein. The rBipD was obtained by affinity chromatography using His Trap column, then mixed with Fredrick's adjuvant to immunize BALB/c mice by intraperitoneal injection in order to obtain anti-rBipD polyclonal antibodies. The immunoreactivity of rBipD was detected by Western blot assay using rabbit anti-melioidosis serum and the serum from melioidosis patients. The immunogenicity of rBipD was evaluated using Western blotting and immunofluorescence staining. Finally, rBipD was used to establish an indirect ELISA to detect serum antibodies of clinical melioidosis patients. Results The recombinant plasmid pET-28a-BipD was successfully constructed and transformed into E.coli BL21 (DE3) to induce rBipD expression with IPTG treatment. The obtained rBipD had a relative molecular weight of 36×103 and a purity of 95.4%, and had good immunogenicity and immunoreactivity. It could induce the production of specific antibodies after immunizing mice, and mouse polyclonal antibodies against rBipD were prepared with the titer of 1∶512 000. rBipD of 5.0 μg/mL produced specific immune response with the serum of melioidosis patients, but had no specific reaction with the serum of tuberculosis patients, with statistical difference (P < 0.01). Conclusion rBipD with immunological activity is successfully prepared and purified, and its polyclonal antibodies are also developed, which provide a good tool for clinical immunological diagnosis and study of immune mechanism of B. pseudomallei infection.

Keywords

• burkholderia pseudomallei
• bipd protein
• recombinant expression
• polyclonal antibodies preparation