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Expression and Immunogenicity of Staphylococcus.aureus Clumping Factor A

Author: FengZuo
Tutor: CuiYuDong
School: Heilongjiang Bayi Agricultural University
Course: Preventive Veterinary Medicine
Keywords: Staphylococcus aureus Clumping factor A Adhesin
CLC: R378.11
Type: Master's thesis
Year: 2008
Downloads: 56
Quote: 1
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Abstract


Staphylococcus aureus is an important pathogen that causes a wide spectrum of infections. S. aureus colonizes and intrudes host by surface adhesins adhering to the extracellular matrix. At early infection phase, one member of adhesins—clumping factor A (ClfA) exerts significant role in this process. Previous studies showed that ClfA is likely to be a good immunogen. For the sake of understanding immunoprotection of ClfA against different S. aureus strains, ClfA was expressed and used as immunogen to immunize mice in this experiment. Then the mice were challenged with different S. aureus strains, and immunoreaction of cytoimmunity and humoral immunity were inspected.clfa genes of S. aureus strains Newman, Wood46 and BMSA/855/23-1 were amplified by PCR, and were introduced into pMD18-T vector subsequently. The recombinant vectors, were sequenced. The nucleotide sequences of clfa were compared with one published previously in GenBank. Then, pQE30/clfa recombinant plasmid was constructed by inserting clfa gene from Newman strain into pQE-30 vector. The recombinant plasmid was transformed into E. coli strain M15 (pREP4), and was induced with IPTG to express recombinant ClfA fusion protein,The expression of the recombinant ClfA protein was detected by SDS-PAGE and analized with western blot using sera from the mice immunized respectively with the recombinant ClfA protein and inactived S. aureus. Then, 120 healthy mice were separated four groups, and respectively immunized with the recombinant protein at 50μg, at 100μg, inactived S. aureus at 2×108 CFU and placebo. Boosting immunization was implemented after primary immunization for three weeks. Two weeks after boosting immunization, each group of the immunized mice was challenged with the S. aureus strain Wood46 (3×109CFU/per mice) and BMSA/855/23-1 (1×109CFU/per mice) separately. At same times, blood samples were drawn every week from three mice in each group since the primary immunization, and the antibody titres and the concentration of IFN-γ、IL-2 and IL-4 cytokines in mice sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA).The results of gene cloning and sequencing were that the clfa gene of S. aureus strain Newman was successfully amplified and cloned, the nucleotide sequences of clfa shares 99.8% consistency with the published one and 99.6% homology with Wood46 and BMSA/855/23-1, those suggested that the ClfA gene was highly conservative. SDS-PAGE results showed that the recombinant ClfA was expressed correctly in E. coli M15 (pREP4), the high antigenicity was comfirmed by Western Blot. The antibody level in sera from mice immunized at 50μg, 100μg protein and whole cell groups reached its peak at two weeks after boost immunization (1:65000, 1:150000, 1:4200). The concentration of IL-4, IL-2, IFN-γin ClfA immuno-groups mice sera increased significantly (p<0.05) at day 14 after boost immunization, while the concentration of those in whole cell immuno-group mice sera did not increase significantly (p>0.05) compared with the control. In a week after challenge, the livability of Wood46 challenged mice is 50% in 100μg protein immuno-group, 12.5% in 50μg protein immuno-group, 0 in inactived S. aureus immuno-group respectively; the livability of BMSA/855/23-1 challenged mice is 37.5% in 100μg protein immuno-group, 0 in 50μg protein immuno-group, 0 in inactived S. aureus immuno-group respectively. The results indicate that the recombinant ClfA protein has high immunogenicity and can also protect against S. aureus challenge to some extent. These results can provide some reference for further studies on and development of ClfA vaccine in the prevention of Staphylococcus aureus infection.

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CLC: > Medicine, health > Basic Medical > Medical Microbiology ( pathogenic bacteriology,pathogenic microbiology ) > Pathogenic bacteria > Cocci > Staphylococcus
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