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Prophase Research on the Genetic Engineering Combined Vaccine of Anthrax and Plague

Author: RenJun
Tutor: ChenZuo;LiJianMin
School: PLA Military Academy of Medical Sciences
Course: Microbiology
Keywords: Anthrax Plague Ferment Purification Combined vaccine
CLC: R392
Type: PhD thesis
Year: 2009
Downloads: 128
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Abstract


Anthrax and plague are all natural foci of deadly infectious diseases, both the causative agent of the disease anthrax Bacillus and Yersinia bacteria is extremely important bioterrorism agents / biological warfare agents. The United States, the former Soviet Union, Japan and other countries have made of Bacillus anthracis and Yersinia pestis biological weapons, the United States in 2001 mailed anthrax terrorist attacks once again reminds us that moment can not be complacent bioterrorism, our country there is a big range anthrax and plague natural foci, wartime are likely to also face the threat of anthrax and plague. In recent years, with the extensive use of antibiotics, the emergence of multi-drug resistant strains of anthrax and plague prevention is even more important. Vaccines for both diseases at home and abroad has been listed anthrax live attenuated vaccine, anthrax supernatant was adsorbed vaccine, the plague inactivated whole bacteria dead vaccines, of plague live attenuated vaccine, but there are side effects and other shortcomings. The current the rPA based build new recombinant subunit anthrax vaccine, the RF1 and RV build new recombinant subunit plague vaccines have entered clinical trials. Clear Bacillus anthracis and Yersinia pestis protective antigen induced immune humoral immune to develop anthrax, plague genetic engineering combined vaccine can simplify the immunization program, to increase user compliance, reduce production costs and saving health resources will respond to anthrax and plague biological warfare / bioterrorism, major infectious diseases and public health emergencies to provide protection. The purpose of this study is the use of genetic engineering technology to prepare plague protective antigen to evaluate the effect of anthrax and plague subunit vaccine combined immunodeficiency, and lay the foundation for joint development of vaccines for anthrax, plague genetic engineering. Prokaryotic expression system has a clear genetic background, simple technology, the production cycle is short, simple culture conditions, is one of the most commonly used exogenous protein expression system. Previously, our laboratory has been successfully expressed in E. coli expression system and purified anthrax protective antigen PA. Taking into account the F1 and V from prokaryotic cells, the molecular weight is relatively modest, the first F1 and V antigens highly expressed in E. coli system research. F1 antigen highly hydrophobic, and thus the expression and purification of a challenge. Previous studies have reported that V antigen purified fusion GST-tag affinity purification, and then remove the label. We consider the non-tagged protein compared to the tagged protein purification steps are more complex, but do not consider the label removed and finished in the remnants of a label or protease, easier for the quality control of the finished product. After several attempts, the final choice of using the pET32a () expression vector in E. coli BL21 (DE3) the unlabeled F1 and V antigen, results show that the the unlabeled F1 and V efficient expression. Subsequent shake flask process zoom back 5 times to 14L bioreactor, then zoom back 5 times to 42L bioreactor successful pilot fermentation process optimization. On this basis were cubic fermentation trial, the results show the stability of the fermentation process, with good repeatability. Then take full advantage of the various properties of the protein separation and purification of target protein. In the V antigen the purification process, the first to the hydrophobic nature of the V antigen of the V antigen is captured from the supernatant after the split bacteria. Then use the V antigen when the pH value is higher than the isoelectric point of the negatively charged nature, will capture the V antigen was further purified by weak the DEAE anion column chromatography using gel filtration chromatography to give the purity gt; 95% of the V antigen. Western blot results show that the purified V antigen in the presence of monomer and dimer in two forms. The study showed that the F1 antigen under physiological conditions to easily aggregate to form polymer, and a multimeric form the F1 antigen monomers F1 antigen has better immunogenicity and protective effect. In this study, using the method of salting F1 antigen preliminary separation and purification, followed by gel filtration chromatography for further pure and exchange buffer, results purity gt; 95% of the F1 antigen. Protein N-terminal amino acid sequence determination is fully consistent with the expected results. On this basis, the use of proteins, aluminum hydroxide adjuvant adsorbed and immunogenicity studies in mice, guinea pigs and rabbits were observed humoral immune response to the antigen, and compare the combined immunization group and separate immunohistochemistry The difference between the humoral immune response. The immunogenicity results in three animal models, the joint the immunohistochemical and separate immunohistochemical compared with similar humoral immune response after one to two weeks after the initial immunization the animal serum can be detected to the corresponding antigen-specific IgG antibody , strengthen the immune reached a high level. The ISOTYPE analysis results show the, mice of immune RPA, the RF1 rV does not change the antigen specificity of the IgG1/IgG2a ratio, antibody reaction tend to Th2 type; mouse long-term immunogenicity studies have shown that antibody titers to maintain more than one year long , indicating that the combination vaccine has a good long-term results. Antigen immunogenicity data, select the anthrax sensitive animal rabbit model for the study of the protective effect of the anthrax attacks, Plague sensitive animal model mice Yersinia pestis attacks the protective effect. Results show the combined immunization, rabbits can resistance subcutaneous 80LD50 on Bacillus anthracis virulence attack, while the adjuvant control group, rabbit all died in 6 days. Mouse model results show that combined immunodeficiency mice resistant to the subcutaneous attack up to 60,000 MLD plague bacteria, adjuvant control group all died within 4 days. In both animal models, combined immunodeficiency survival rate / survival time of not less than immunohistochemistry alone. In this study, prepared by genetic engineering techniques, the protection of the plague antigens F1 and V, combined with the anthrax protective antigen PA, the use of aluminum hydroxide adjuvant adsorption, preliminary evaluation of antigen combined immunodeficiency mice, guinea pigs, rabbits immune effect and protective effect. The results show that the combined immunization can effectively protect the virulence of Yersinia pestis and Bacillus anthracis attacks. In addition, the antibody titers after immunization in mice to maintain more than one year, indicating that the combination vaccine has a good long-term effect.

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