Mink Outer Membrane Protein OprF/I Of Pseudomonas Aeruginosaexpressed In E.Coli Efficiently And The Establishment Of ELISA Detection Method

Pseudomonas aeruginosa is widely distributed in nature and it may lead to various infections. High mortality and resistance to antibiotics pose great burdens to clinical treatment. Vaccine has become an effective measure to prevent infections caused by P.aeruginosa. Based on reported Opr F and Opr I genes of P.aeruginosa, this study used PCR technology to amplify the fusion gene Opr F190-342-Opr I21-83 and performed prokaryotic expression to obtain a fusion protein of 25.2ku. This purified protein was used as the antigen to establish indirect ELISA of P.aeruginosa Opr F/I protein. Laboratory mice were immunized with mixtures of different adjuvants and purified Opr F/I protein. The level of specific antibodies in mice serum was then detected.The main research contents include:1. Based on reported P.aeruginosa Opr F gene（Gen Bank: JX040481.1） and Opr I gene（Gen Bank: X58714.1）, Opr F190-342, Opr I21-83 and fusion gene Opr F190-342-Opr I21-83 with sizes of 495 bp, 207 bp and 663 bp respectively were cloned using PCR technology.2. Prokaryotic expression and purification of P.aeruginosa outer membrane proteins. Based on cloned gene fragments Opr F, Opr I and Opr F/I, recombinant expression vectors p Cold II-Opr F, p Cold II-Opr I and p Cold II-Opr F/I were constructed and their expressions were induced in E.coli. Recombinant proteins Opr F（19ku）, Opr I（8.93ku） and Opr F/I（25.2ku） were obtained by AKTA purifier system. Results of western blot analysis indicated that the recombinant proteins Opr F, Opr I and Opr F/I possessed good reactionogenicity.3. Establishment of P.aeruginosa indirect ELISA. Purified proteins Opr I and Opr F/I as well as JL08 and DL15 strains treated by ultrasonication were used as antigens to establish ELISA. Comparison results showed that using purified proteins as antigens had a better sensitivity for mice serum infected by P.aeruginosa than the whole cell antigen.4. Purified recombinant protein Opr F/I was respectively mixed with adjuvants Al（OH）₃ and ginsenoside. Laboratory mice were immunized and serum specific antibodies were detected. The results demonstrated that antibodies were generated one week after immunization and the persistent period was about 56 days. Different dosages of ginsenoside addition led to different immune effects. Al（OH）₃ showed a prominent advantage in the initial stage of immunized group over other groups.