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Study on Bayesian Kirk body immune protein group

Author: XiongXiaoLu
Tutor: WenBoHai
School: PLA Military Academy of Medical Sciences
Course: Microbiology
Keywords: Coxiella burnetii Q fever immunoproteomics protein microarray protective antigens
CLC: R392.1
Type: PhD thesis
Year: 2011
Downloads: 55
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Coxiella burnetii, the etiological agent of Q fever, is highly infectious to both human and livestock. Human Q fever is generally acquired via the respiratory route by inhalation of infectious aerosols produced by domestic livestock. The common symptoms of human Q fever are pneumonia, hepatitis, and endocarditis. C. burnetii is recognized as an important agent of bio-warfare/bio-terrorism due to its high pathogenic and ability to resist the adverse environments and physical or chemical factors.The clinical diagnosis of Q fever is mainly based on serological tests including indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and complement fixation (CF) tests. However, the antigens currently used in these tests are coxiella organisms which are difficult and hazardous to culture and purify. Therefore, these tests are of limited use for large-scale application. Inoculation of vaccines against Q fever is an effective way to prevent the human infection caused by C. burnetii. The Q fever vaccine derived from inactivated C. burnetii whole-cells has the capability to induce protective immunity against Q fever; however the strong side effects elicited by its immunization limit its widespread use. Efforts have been underway to develop a safer, more effective new-generation vaccine against Q fever. In this study an immunoproteomic study of C. burnetii Xinqiao strain was conducted by probing the proteins of C. burnetii whole cells separated by 2-D electrophoresis with infection sera of Q fever. The proteins identified will be cloned and expressed in vitro, then evaluated for their immunogenicity and protectivity to serve as candidates for diagnostic marker and multivalent vaccine against Q fever.C. burnetii Xinqiao strain was propagated in embryonated eggs and purified by a renografin density centrifugation. BALB/c mice were injected intraperitoneally with C. burnetii Xinqiao strain. The mice were killed on weeks 1, 2, 3, and 4 post-infection, respectively. The sera from mice sacrificed were collected and tested for specific antibody and the liver, spleen, and lung tissues were detected by a real-time quantitative PCR specific for C. burnetii. A large amount of C. burnetii was found in liver, spleen, and lung tissues post-infection and the highest level was detected on week 1 post-infection; the amount of C. burnetii in spleen tissues was significantly higher than that in liver or lung tissues. The amount of C. burnetii in tissues significantly reduced on week 2 post-infection. However, the levels of specific antibody detected by indirect immunofluorescence assay rose with the infection progress.The total protein of C. burnetii purified were separated by 2-dimensional electrophoresis and analyzed with sera from experimentally infected BALB/c mice and Q fever patients by immunoblot assay. Then the immunoreactive proteins were identified by mass spectrometry. As a result, 0, 4, 9, and 14 of the coxiella proteins were recognized by the mouse sera obtained at 1, 2, 3, and 4 weeks post-infection, respectively. Among these recognized proteins, 4 proteins, GroEL, Mip, OmpH, and Com1, were strongly recognized by sera of mice infected with C. burnetii. In addition, 15 of the coxiella proteins were recognized by sera from two patients with acute Q fever. Among these immunoreactive proteins, 9 proteins were recognized by both the mouse and human sera.The genes encoding the 20 immunoreactive proteins were amplified from the genomic DNA of C. burnetii. The PCR products were ligated into the prokaryotic expression plasmid pET32a/pQE30. Except RspB, the 19 recombinant immunoreactive proteins were successfully expressed in E. coli cells and purified by Ni-NTA agarose, respectively. The 19 recombinant proteins were used to fabricate a protein microarray. The protein microarray was probed with sera from mice infected with C. burnetii and 0, 16, 19, and 18 of the recombinant proteins on the microarray were recognized by mouse sera collected on weeks 1, 2, 3, and 4 post-infection, respectively. Four proteins, GroEL, Mip, OmpH, and Com1, particularly GroEL, were strongly recognized by the mouse sera. Then the protein microarrays were probed with 56 sera from Q-fever patients and 25 sera from healthy persons. Proteins were considered seroreactive if the signal intensity in probing with one patient’s serum were higher than that the mean plus 2 times the standard deviation in probing with normal sera. GroEl, YbgF, RplL, Mip, OmpH, Com1, and Dnak were positively recognized by more than 40% of the sera of patients with acute Q fever, suggesting that the 7 protein antigens are good candidates for a new Q fever serodiagnostic assay.Mouse bone marrow dendritic cells (BMDCs, CD11c+) were isolated from bone marrow of BALB/c mice. After 6 days of culture, BMDCs were stimulated for 24 h with recombinant proteins Com1, GroEL, Mip, and YbgF, respectively, and C. burnetii whole cell antigen (WCA), E. coli LPS, TrxA encoded by pET32a, or elution buffer were used as parallet controls of pulsing BMDCs. The resulting populations of BMDCs were analyzed by flow cytometry to determine the expression of surface molecules. Antigen-treated BMDCs exhibited substantially increased expression of surface molecules and the expression levels of surface molecules were higher than that of elution-pulsed BMDCs. The pulsed BMDCs were intraperitoneally injected into BALB/c mice. On day 1, 7, 14 post-transfer BMDCs, mice were intraperitoneally challenged with C. burnetii, respectively. Mice were sacrificed on day 7 postchallenge and their spleens were harvested for detection of C. burnetii burden in the mice by a quantitative PCR analysis. Compared with mice receiving no BMDCs pulsed (negative control), mice receiving BMDCs pulsed with WCA, Com1, or Mip exhibited significantly lower coxiella burden, while mice receiving BMDCs pulsed with GroEL, YbgF, TrxA, or LPS displayed similar high levels of coxiella burden. These results suggest that Com1 and Mip are potent protective antigens of C. burnetii and may be considered as candidates of a subunite vaccine of Q fever.The antigen-pulsed BMDCs were cocultured with the isolated CD4+ and CD8+ T cells, respectively. Phenotypic molecules and intra-cellular cytokines of CD4+ and CD8+ T cells were analyzed by flow cytometer. CD4+ and CD8+ T cells from mice immunized with WCA-, GroEL-, Com1-, or Mip-pulsed BMDCs were efficiently activated with significant higher expressison of CD69 than that from mice immunized with elution-pulsed BMDCs. While the T cells from mice immunized with WCA-, Com1-, or Mip-pulsed BMDCs exhibited a significantly higher level of IFN-γcompared with that from GroEL-pulsed BMDCs, which suggests that protection immunity offered by BMDCs stimulated with the protective antigens is associated with proliferation and activation of T cells that are more potent to produce IFN-γto activate macrophages for killing of the intracellular pathgens.

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