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Study about Influence on Immunogenicity of Molecular Adjuvant C3d to Major Subunit FimA of Type Ⅰ Fimbriae in Salmonella Enteritidis

Author: ZhangWeiJuan
Tutor: ZhuGuoQiang
School: Yangzhou University
Course: Preventive Veterinary Medicine
Keywords: SE FimA The FimA-mC3d_n fusion protein Immunogenicity Immune protective effect
CLC: R378
Type: Master's thesis
Year: 2010
Downloads: 43
Quote: 1
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Abstract


Salmonella enteritidis (Salmonella enteritidis, SE) can not only cause livestock disease, is also one of the main pathogens of food poisoning in humans, its widely popular not only caused great economic losses to the society, but also a serious threat to people's life and health, human public health hazard. The study found that Salmonella enteritidis can adhere to and invade a variety of cells, this ability is considered to be its major pathogenic the pili as bacterial adhesion and invade host cells virulence factors, their pathogenic Sexually has a vital role. Salmonella enteritidis type Ⅰ, Ⅱ, Ⅲ, Ⅳ, SEF14 and SEF18 major fimbriae, which the majority of Salmonella enteritidis expression of type Ⅰ fimbriae, FimA type I fimbriae structural subunit, which encodes gene fimA. C3d complement C3 molecule can no longer be the smallest fragment of the protease hydrolysis When C3d and connected to the antigen molecule directly with antigen presenting cells of the CR2 receptor binding, reduce the threshold of the activation of B lymphocytes, thereby enhancing the antigenic molecules of the immune immunogenicity. Therefore, C3d novel molecular adjuvants, increasing cause for concern. Succession Reports have confirmed the C3d molecular adjuvant effect, but these are protein and DNA vaccines based on the expression of eukaryotic expression vector, on the the C3d molecule expression of prokaryotic vector protein immunogenicity, there is no relevant reports. Subunit encoding gene fimA expression of prokaryotic vector protein immunogenicity explore C3d molecules, this study selected the type I fimbriae of Salmonella enteritidis main purpose gene, build the pCold-fimA and containing different copy mC3d pCold-fimA-mC3dn The recombinant plasmid. Main building: fimA fragment was amplified by PCR from the standard strain of Salmonella enteritidis SD-2 chromosome, PCR products were purified, connecting T vector and sequenced to verify the correct target gene the fimA insert the expression vector pCold-TF build the recombinant plasmids pCold-fimA, the build good recombinant plasmid pUC-mC3d (single copy) has the pUC-mC3d2 (2 copies) and pUC-mC3d3 (3 copies) restriction endonuclease digestion, the digestion product by agar After the sugar gel electrophoresis identified by agarose gel DNA extraction kit recycling by the above-mentioned of different copy mC3d the of gene fragments mC3d, mC3d2 and mC3d3, respectively inserted into the building on the good recombinant plasmid pCold-fimA get fimA series different The the the the copy mC3d the recombinant plasmid pCold-fimA-mC3d, pCold-fimA-mC3d2 and pCold-fimA-mC3d3. Based on the above recombinant plasmid construct induced by IPTG 15 ℃ for 24 h to obtain high-level expression of soluble FimA protein and the FimA-mC3dn series a different copy mC3d the fusion protein using Novagen Company immobilized Ni2 affinity chromatography kit purified recombinant protein identified by SDS-PAGE, and 70 kDa, respectively, 100kDa at 130 kDa and 160 kDa with Western-blot results appear consistent with the expected size of the specific article, of Salmonella enteritidis I pili multi-resistant identification by the restructuring protein, suggesting that it has good immunogenicity and reactogenicity. To further explore the impact of C3d molecules FimA immunogenicity, expression of the the purified soluble FimA, FimA-mC3dn fusion protein immune BABL / c mice. Immunization program: the first free, soluble of FimA of FimA-mC3d of FimA the-mC3 d2 and of FimA-mC3d3 nude antigen immune 0.1μg, 1μg and 10μg 3 immune-dose group; each protein sub-interval of 35 d II Stock were with Freund's complete adjuvant emulsified FimA immune immunization dose of 10μg. Immune process weekly lancing serum separated by indirect ELISA for detection of antibody titer change. Free one week after two SD-2 experimental mice with standard strains of Salmonella enteritidis challenge attack toxic dose of 10 cfu, and then observed the death of the mice in each group. Protein experimental results show that when the immunization dose 0.1μg and 1μg anti FimA antibody titers were no significant differences between the FimA immune the group and the FimA-mC3dn immune group. Immunization dose of 10μg FimA immunohistochemistry, FimA-mC3d2 the FimA-mC3d3 immunohistochemistry antibody titers were increased 2-fold and 4-fold; but the FimA-mC3d with FimA difference is not obvious; When the immunization dose 10μg FimA-mC3d the FimA-mC3d3 the antibody titer and FimA-mC3d2, the antibody titer increased 4-fold and 2-fold, respectively. The challenge protection experimental results show that, in the immunizing dose of 10μg FimA immune protection of the group was 50% (2/4), while the group of the FimA-mC3d2 and the FimA-mC3d3 immune protection rates were 75% (3 / 4) and 100% (4/4). Collectively, these results show that C3d expression of prokaryotic vector Immunogenicity enhanced further confirmed the C3d molecule immunoadjuvant role and provide the basis for the C3d control in prokaryotic system, in-depth study of the immunogenicity and Salmonella.

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