Prokaryotic expression, polyclonal antibody preparation and immunoprotection potential of Pseudomonas aeruginosa outer membrane protein OprH / 第二军医大学学报

ABSTRACT

Objective To lay a foundation for the industrial fermentation and vaccine development of Pseudomonas aeruginosa (P. oeruginosa) outer membrane protein OprH. Methods The OprH expression strain was obtained by molecular cloning. The culture condition and optimal expression of the experiment was obtained by the method of orthogonal design. OprH was purified by gel slice strategy and was used to immunize mice to prepare the polyclonal antibody. The antibody titer and specificity were detected by ELISA and Western blotting analysis, respectively. Mice were immunized by OprH protein and infected with P. aeruginosa, and the immune protection of OprH was detected. Results OprH recombinant vector were digested and sequenced, and the results confirmed the correct construction; and OprH expression and purification of strip size agreed with the prediction. The optimal culture condition was as follows rotation rate was 230 r/min, glucose concentration was 0%, and the medium volume was 50 mL. The optimal inducing expression condition of OprH was as follows isopropy-β-D-thiogalactoside final concentration was 0.3 mmol/L, strain D600 value was 0.8, inducing temperature was 32℃, and inducing time was 3 h. The OprH antibody titer was 11 600 as detected by ELISA, and Western blotting analysis proved that the antiserum had good specificity. Mice specific immune was activated by OprH, and immune protection rate for mice against P. aeruginosa infection was 46.15 %, which had significant differences compared with the control (P<0.05). Conclusion We have successfully cloned OprH expression vector, purified OprH, prepared the polyclonal antibodies of OprH. It is confirmed that OprH protein has significant immune protection against P. aeruginosa, and the culture and induction conditions of the recombinant OprH have been obtained.