The Preliminary Experimental Screening Of Neisseria Gonorrhoeae’s Outer Membrane Proteins Based On The Reverse Vaccinology

ObjectivesTwo kinds of outer membrane protein and one kind of none-outer membrane protein were selected from the Neisseria gonorrhoeae’s potential outer membrane protein that had been predicted by bioinformatics software, in order to clone and express the recombinant protein which was used to immune mice for obtaining the antiserum of the recombinant protein. The antigen of predicted membrane protein was used for surface’s location of bacteria and on the preliminary experimental study of immunogenicity and immune reactivity, so as to discuss the feasibility of reverse vaccinology combined with experimental method for screening vaccine candidates of the Neisseria gonorrhoeae, and to offer a new idea for the research of gonococcal vaccine.Methods1Clone and expression of the potential outer membrane protein: a series of outer membrane proteins had been predicted and analyzed from the whole genome database of Neisseria gonorrhoeae of previous research, in which two kinds of outer membrane proteins（YP₂07398.1 and YP₂07701.1）and one kind of none-outer membrane protein（YP₂07727.1） were selected randomly, with the best primers designed by the Premier 5.0 software, three segments of target gene were amplified by polymerase chain reaction（PCR） from the standard strains（ATCC 49226） genome of Neisseria gonorrhoeae, then, the recombinant plasmid of three kinds of genes was constructed with p GEX4T-1as its plasmid vector, and then the recombinant proteins was induced to be expressed in host bacteria E.coli BL21, and finally, the expression of the recombinant protein by SDS-PAGE was analyzed after optimizing the expressive condition of recombinant protein.2Purification of recombinant proteins: After the bacteria was broken by ultrasonic wave, the bacterial precipitation was collected by centrifugation.Then the Triton X-100 and urea were used to wash the inclusion,and urea were used to dissolove the inclusion which was washed, renatured and purified in the following steps, meanwhile, the method of cutting polyacnylamide gel was also used to get the purified protein.3The preliminary research of the immune reactivity, immunogenicity andbacterial membrane surface’s location of the recombinant proteins:（1） the method of ethanol inactivation was applied to inactivate the standard strains（ATCC 49226） of Neisseria gonorrhoeae to prepare immunogen to immune rabbit and obtain the immune serum which would be taken as the primary antibody. Then, the Western Blot technique was adopted to detect the antigenicity of the purified recombinant protein.（2） The preparation of recombinant protein antiserum: each recombinant protein with dose of 50μg was mixed with freund’s adjuvant to immune BALB/c mice. The immunization was carried out for 2 times in total and at intervals of 2 weeks.The immune serum could be harvested in the following week after the last immunization.（3） The bacteria surface’s location of outer membrane protein antigens: the whole-cell antigen of 96-well plate prepared by the standard strains（ATCC 49226） of Neisseria gonorrhoeae was treated with 75%alcohol, Then, taking the immune serum of mice as primary antibody and the Goat anti Mouse Ig G with enzyme marker as secondary antibody, the serum antibody titer could be detected by ELISA, so as to evaluate the immune reactivity of the recombinant proteins and binding capacity of antibody and protein of cell surface, as well as to determine preliminarily the immunogenicity of the predicted outer membrane protein and whether it is located on the surface of Neisseria gonorrhoeae.Results1The recombinant prokaryotic expression plasmid of three genes of outer membrane protein of Neisseria gonorrhoeae was successfully constructed as the followings:PGEX4T-1-YP₂07398.1,PGEX4T-1-YP₂07727.1,PGEX4T-1-PGEX4T-1-YP₂07701.1, SDS-PAGE showed that three recombinant protein were expressed in the form of inclusion body by IPTG induction, the best expressional condition has been concluded as these: The YP₂07398.1protein: 37 ℃, 2.0 mmol/L IPTG induced 5 hours; The YP₂07701.1protein:37℃ and 2.0 mmol/LIPTG induced 1 hour; The YP₂07727.1 protein:37℃, 0.5 mmol/L IPTG induced 4 hours.2Western Blot analysis shows this: the candidate outer membrane protein antigen can be identified by anti-neisseria gonorrhoeae’s polyclonal antibody.3The analysis of Western blot showed: the antigen of candidate membrane protein could be identified by the polyclonal antibody of Neisseria gonorrhoeae’s resistance.4After successfully obtaining the purified protein, the inclusion bodies was washed by the Triton X-100 and urea, and the washed inclusion body would be dissolved by the solution liquid of inclusion body. It was found that three recombinant proteins were not sufficiently dissolved. Then, the methods of gel cutting, electric elution and dialysis were adopted, and the crude extract of recombinant proteins could be obtained.5Obtaining successfully three kinds of recombinant protein antiserum, the results of the indirect ELISA detection reveals this: the immune serum antibodies from the Neisseria gonorrhoeae’s whole-cell antigen immune group, the YP₂07701.1 protein immune group and the YP₂07398.1 protein immune group, are able to bind specifically with Neisseria gonorrhoeae’s whole-cell antigen, and the antibody titer of the YP₂07701.1 protein immune group was higher than the YP₂07398. 1 protein immune group, while the immune serum antibodies of the YP₂07727. 1 immune group couldn’t combine obviously with whole-cell antigen of Neisseria gonorrhoeae.Conclusion1Three prokaryotic expression vectors were successfully constructed and expressed in escherichia E. coli BL21, which laid foundation for the presente research and the subsequent Neisseria gonorrhoeae other research;2After Indirect ELISA detection of the recombinant protein antiserum and bacteria Neisseria gonorrhea’s whole-cell antigen, it was found that as followings: The two recombinant protein predicted as the outer membrane protein could be reacted with Neisseria gonorrhoeae’s whole-cell antigen, and it’s antibody in the recombinant protein serum was higher while the protein predicted as the none-outer membrane protein couldn’t be reacted with Neisseria gonorrhoeae’s whole-cell antigen. These results preliminarily showed that the two recombinant proteins were located on the cell surface of Neisseria gonorrhoeae, which has good immunogenicity and immune reactivity.3This research provides experimental datas and methods for the experimental screening of candidate outer membrane protein, and it also provides a new thought for the research of gonococcal reverse vaccine candidates.