Cloning,expression And Antigen Immune Protection Study Of A Outer Membrane Protein Of Severe Infective Acinetobacter Baumannii

Objective:Through reverse vaccinology strategy and bioinformatics analysis, from the Acinetobacter baumannii genome-wide screening,acquireed a number vaccine candidate antigens of Acinetobacter baumannii, A1S₀115 is an unknown function of acinetobacter baumannii outer membrane proteins,which has good conservative and specificity, in order to study the protein of immunogenicity and immune protection, explore the protein as the possibility of the potential vaccine antigens of Acinetobacter baumannii, this study through gene cloning and expression of Acinetobacter baumannii A1S₀115 extracellular protein zone（A1S₀115A） active fragments as vaccine candidate antigens, the antigen in mice with Acinetobacter baumannii infection in animal models,to evaluate immune protection, at the same time for the candidate antigen induced immune protection mechanism is studied. looking forward to lay a foundation of experimental basis and theoretical guidance for the new and effective genetic engineering vaccine of molecular design, the antigen, and subsequent recombinant subunit vaccine research.Method:1. Extract bacterial genome from Acinetobacter baumannii international standard strains 17978 as the PCR template, after bioinformatics analysis, by PCR amplification A1S₀115 extracellular region active fragments（named: A1S₀115A）, at the same time introduce an enzyme loci of BamH I and Not I. After double enzyme digestion of target gene,the recycling products linked to pGEX- 6 p- 2 expression vector,convert into Escherichia coli XL-1 blue strains, under the induction of IPTG,the Escherichia coli express GST fusion protein, the PP enzyme pure the protein with a GST fusion labels, then purification with the chromatography, endotoxin fine purification, the final product by SDS-PAGE electrophoresis analysis.2. To evaluate the candidate vaccine antigens A1S₀115A immunogenicity, by ELISA in the different time points after immune detection the specific antibody level of the candidate vaccine antigen and its subtypes, and draw the antibody growth curve3. To evaluate the candidate vaccine antigens A1S₀115A immune protection, through abdominal cavityinfectionof Acinetobacter baumannii standard strains 17978, establish BALB/c mice sepsis animal model. BALB/c mice immune with a candidate protein antigen and AL（OH） 3 immune adjuvant, and histidine buffer and AL（OH） 3 adjuvants as control group. Two week after the last immune mice infected with Acinetobacter baumannii LD80 dose 4.2 x 108 CFU, 7 days after infection monitor the condition of mice. At the same time monitor organs bacterial burden and pathological damage.4. In order to evaluate candidate protein immune protection mechanism, detect the specific antibody in vitro mediated opsono-phagocytosis, evaluation A1S₀115A specificity antibodies in vivo passive immune protection effect. While detecting the immune mice after infected with acinetobacter baumannii in the serum proinflammatory factor（IL-1 β, gm-csf） and cytokines in the spleen cells（IFN-γ, IL- 4） level.Results:1. Successfully Constructe the expression vector plasmid pGEX-6p-2-A1S₀115A, by IPTG induction, candidate vaccine proteins expressed in soluble form and its purification is efficient, SDS-PAGE analysis of the protein molecular weight in line with expectations.2. Candidate vaccine antigens A1S₀115A D0, D7, D14 immune program immune BALB/c mice, mice produced specific humoral immune response, the antibody titer induced in 25, 6000, specific antibody subtypes are mainly composed of IgG1. In the last 3 months after immune detection time, A1S₀115A specific antibody level is gradually increased and stable, has a relatively good immune persistence.3. The establishment of a stable BALB/c mice sepsis animal model. Mice immuned with candidate A1S₀115A protein antigens, lethal dose Acinetobacter baumannii infection, compared with control group, the survival rate of the immuned mice improved obviously; after immuned with the candidate protein antigent, the bacteria burden in the spleen, kidneys, heart, liver, lungs was reduced at a certain extent, alleviate the pathological inflammatory damage at the same time, clinical scoring in mice immune group was obviously higher than that of control group.4. Antibodies activity in vivo and in vitro experiment and function test results show that the specificity of the purified A1S₀115A antibody in vitro has obvious opsono-phagocytosis effect, in Acinetobacter baumannii infection mice has good passive immune protection effect. Immunity of mice after infected with Acinetobacter baumannii cytokines detection results show that the vaccine group spleen cells of IFN- γ and IL- 4 expression level is significantly higher than the control group（p < 0.05）, while in the serum proinflammatory factor IL- 1 βand gm-csf expression level, vaccine group than the control group were significantly lower（p < 0.05） comparedMice immuned with Candidate A1S₀115A protein antigens produced a strongConclusion:1. Successful build pGEX-6p-2-A1S₀115A/blue XL- 1 gene engineering bacteria, and the preparation of high purity of Acinetobacter baumannii A1S₀115A candidate vaccine antigens. The antigen immuned mice induced to produce the effective price protective antibody, for a lethal dose of acinetobacter baumannii infection with good immune protection. Indicates that the candidate has good immunogenicity and immune protection.2. Preliminary research of immune protection mechanism show that the immune vaccine candidate antigens can stimulate the body to produce specific protective humoral immune responses and cell immune response, and effectively suppress the Acinetobacter baumannii infection of proinflammatory factor expression, protection mice from sepsis shock and death caused by Acinetobacter baumannii infection.3. This research lay a foundation for new, effective, genetic engineering vaccine of Acinetobacter baumannii for antigens selection, molecular design, immune protection evaluation, for subsequent research and development of Acinetobacter baumannii recombination polyvalent subunit vaccine provides a solid experimental basis and theoretical guidance.