| Plague and anthrax are natural epidemic fulminating infectious diseases that pose grave threats to human’s health and safety. The pathogens of these 2 diseases, Yersinia pestis and Bacillus anthracis, are listed by America as the key pathogens in anti-terrorist and top level class A pathogens. Moreover, there are many areas in China that have a high occurrence rate of plague and anthrax, and occasionally epidemic situations in China have reported in recent years. Whether it’s peace time or at war, there’s risk of facing plague and anthrax at the same time. Therefore, it’s of vital importance to take both civil and military use into consideration while researching the vaccine of plague and anthrax. Especially in recent years, after the appearance of drug-resistant strains caused by overuse of antibiotics, that the precaution of plague and anthrax is becoming more and more important.In this study, flagellin was used as the vaccine adjuvant, the protective antigen of Yersinia pestis, F1 and V, and the neutralizing epitope region 2β2-2β3loop located in the domain 2 of the protective antigen PA in Bacillus anthracis were used as the effective components of the vaccine to study the feasibility of combined plague and anthrax vaccine. Flagellin is the ligand of the Toll-like receptor 5(TLR5), which combines congenital immunity and adaptive immunity by stimulating the production of cell factors and the maturity of dendritic cells to present the adjuvant function. Complete flagellin or its fragments can act as adjuvant, including mucosal adjuvant. The vaccine based on flagellin adjuvant only requires a small dose to activate an effective immune response, and the flagellin still has adjuvant activity in the older immune system. Considering all the advantages of flagellin as vaccine adjuvant, recombinant plague antigen, anthrax epitope recombinant protein and plague and anthrax recombinant fusion protein were established basing on flagellin adjuvant. Basing on the assessment of recombinant plague antigen and anthrax epitope recombinant protein, this paper also discussed the mechanism of the immune response of the combined plague and anthrax vaccine and assessed the effect of flagellin as vaccine adjuvant.In this study, F1 antigen was mutated through gene recombination first to obtain the F1 mut mutant, in which the groove formed by F1 monomer could close its own N-ternimal and form intramolecular complementation, so that F1 could exist as monomer and wouldn’t get together easily. The V antigen was fused to the C-terminal of F1 mut, and then established F1mut-V was cloned to the C-terminal of the 185 th amino acid of flagellin which had deleted the 186th- 397 th amino acid. Then the recombinant fusion protein Fli Cdel-F1mut-V was obtained. The successful expression of the recombinant fusion protein and its ability to combine with F1 and V monoclonal antibodies were validated by Western blotting. The assessment of immunogenicity and immunoprotection was conducted on mouse models. The result of serological detection in immune mice indicated that Fli Cdel-F1mut-V induced high titer anti-F1 and anti-V antibodies; the result of challenge experiments indicated that after the use of recombinant plague antigen Fli Cdel-F1mut-V that taking flagellin as the adjuvant on the mice, it could 100% protect the mice from the attack of 104 CFU plague standard 141 virulent strains.When fighting against bio-terrorism, it’s usually required to handle multiple virulent pathogens at the same time. Developing the combined vaccine of multiple virulent pathogens can prevent 2 or more diseases at the same time, which will significantly reduce the immunization times and save medical resources. In addition, whether peace time or at war, the application of combined vaccine can increase the compliance of inoculators, especially for children. This study conducted exploratory research on the combined plague and anthrax vaccine taking flagellin as the adjuvant. Previous work in our lab has proved that 2β2-2β3loop is the important neutralizing epitope region of anthrax protective antigen PA. Basing on flagellin adjuvant, plague and anthrax recombinant fusion protein Fli Cdel-F1mut-V-PAloop2 and anthrax epitope recombinant protein Fli Cdel-PAloop2 were established in this study. The successful expression of Fli Cdel-PAloop2 and Fli Cdel-F1mut-V-PAloop2 was validated by Western blotting, and the ability of Fli Cdel-PAloop2 and Fli Cdel-F1mut-V-PAloop2 to combine with the specific monoclonal antibody of PA domain 2 was detected by Dot blotting and ELISA. In the serological detection of Fli Cdel-F1 candidate epitopes. The recombinant protein presented consistency in Western blotting and ELISA, which could be considered that the selected peptide sequence was mimic epitope of F2H5. Research on F1 epitope is still some kind of basic exploration, thus its effectiveness is required to be validated by further experiments.In conclusion, this study used a good vaccine adjuvant Fli Cdel, established recombinant plague antigen Fli Cdel-F1mut-V basing on flagellin, validated in a mouse model that Fli Cdel-F1mut-V had good immunizing protection and this recombinant plague antigen has a very good application future. Moreover, anthrax epitope recombinant protein Fli Cdel-PAloop2 and combined plague and anthrax vaccine Fli Cdel-F1mut-V- PAloop2 basing on flagellin were established, and humoral immune responses in both rat and mouse models were induced, the survival time of mouse attacked by anthrax toxin was extended; the adjuvant function of flagellin was proved in rat model. In addition, 2 mimic epitopes of F1 antigen were screened, which provides a certain basis for the research of plague epitope vaccine. immunized rats, both Fli Cdel-PAloop2 and Fli Cdel-F1mut-V-PAloop2 could induce PA specific antibodies, but the antibody level was lower than that induced by r PA; while the level of PA specific antibodies produced in Fli Cdel-F1mut-V-PAloop2 group was generally lower than that in Fli Cdel-PAloop2 group, it’s probably because the functional domain of 2β2-2β3loop region was covered during the folding process of recombinant protein which reduced the ability to induce the body to produce antibodies, or probably influenced by the difference of physical and chemical properties of each component of the combined vaccine. In rat model, the level of PA specific antibodies stimulated by Fli Cdel-PAloop2 and Fli Cdel-F1mut-V-PAloop2 with or without aluminium hydroxide adjuvant has no significant difference, while the level of PA specific antibodies stimulated by Fli Cdel-PAloop2 without adjuvant was second only to that in r PA immunization group, indicating that Fli Cdel itself was a very good adjuvant. It’s detected in mouse model that Fli Cdel-PAloop2 and Fli Cdel-F1mut-V-PAloop2 could induce the production of PA neutralizing antibodies, but the level was lower than that in r PA immunization group, and the mice that produced neutralizing antibodies could have a longer survival time after the injection of anthrax toxin, indicating that it’s not enough to replace the whole PA by only one epitope of 2β2-2β3loop in PA structural domain2, the presence of other neutralizing epitopes is required. In addition, in the mouse model, detecting higher level of F1 and V specific antibodies in the immunized serum with recombinant vaccine Fli Cdel-F1mut-V-PAloop2.In order to avoid small molecular proteins or epitopes are covered by macromolecular proteins structurally in the combined vaccine and affect the effectiveness of each component, neutralizing epitopes of antigens were used to replace the whole antigen to stimulate the reasonable design of the combined vaccine. In this study, the neutralizing monoclonal antibody F2H5 of F1 antigen of plague was used to screen the 12 peptide phage display libraries, and 2 strains of phage monoclones that could combine with F2H5 competing with F1 antigen were obtained, their peptide sequences were FIPEVRDPRYHP and MTLDLPDLRYGF respectively. However, when these 2 peptide sequences didn’t have consensus sequences with F1 antigen in sequence comparison. Later, gene recombination of the selected peptide sequence and Fli Cdel was conducted to obtain the recombinant fusion protein... |
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