牛乳源金黄色葡萄球菌EsxA蛋白的表达及免疫原性分析
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摘要
为分析牛乳源金黄色葡萄球菌(Staphylococcus aureus)EsxA蛋白的免疫原性,构建EsxA-p ET-28a重组表达质粒,重组质粒经诱导表达后进行SDS-PAGE和Western blotting鉴定。用纯化后重组EsxA蛋白免疫小鼠,用间接ELISA检测免疫小鼠血清中的IgG、IgG1和IgG2a水平;免疫小鼠经S.aureus菌株攻击后,检测小鼠肝、脾、肾组织荷菌数和免疫保护率,观察S.aureus菌株攻击后小鼠肝、脾、肾病理组织学变化。结果表明,成功诱导表达了EsxA重组蛋白,该重组蛋白免疫小鼠后血清抗体效价可达1∶900,与对照相比,重组蛋白免疫后可减少小鼠肝、脾、肾组织的荷菌数,减轻这些脏器的病理损伤,对免疫小鼠保护率达75%。上述结果表明,该重组Esx A蛋白具有良好的免疫原性。
To study the immunogenicity of EsxA protein of Staphylococcus aureus, the EsxA-pET-28 a recombinant plasmid was constructed and the expression product was analyzed by SDS-PAGE and Western blotting after the positive recombinant plasmid was induced by IPTG. Mice were immunized with purified EsxA protein and then the IgG, IgG1 and IgG2 a antibody were detected with indirect ELISA. Then the histopathological examination, bacteria loading and immune protection of immunized mice were studied after challenge with S. aureus. The recombinant protein EsxA was successfully induced and expressed. After immunization the EsxA specific antibody titer could reach 1:900. Bacteria loading and pathological damage of liver, spleen and kidney were reduced after immunization with EsxA in the immunized mice. The protection rate of immunized mice was 75%. In conclusion, EsxA protein has good immunogenicity.
To study the immunogenicity of EsxA protein of Staphylococcus aureus, the EsxA-pET-28 a recombinant plasmid was constructed and the expression product was analyzed by SDS-PAGE and Western blotting after the positive recombinant plasmid was induced by IPTG. Mice were immunized with purified EsxA protein and then the IgG, IgG1 and IgG2 a antibody were detected with indirect ELISA. Then the histopathological examination, bacteria loading and immune protection of immunized mice were studied after challenge with S. aureus. The recombinant protein EsxA was successfully induced and expressed. After immunization the EsxA specific antibody titer could reach 1:900. Bacteria loading and pathological damage of liver, spleen and kidney were reduced after immunization with EsxA in the immunized mice. The protection rate of immunized mice was 75%. In conclusion, EsxA protein has good immunogenicity.
引文
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[19]Korea CG,Balsamo G,Pezzicoli A,et al.Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells.Infect Immun,2014,82(10):4144-4153.
[20]Wang YN,Hu M,Liu Q,et al.Role of the ESAT-6secretion system in virulence of the emerging community-associated Staphylococcus aureus lineage ST398.Sci Rep,2016,6:25163.
[21]Sundaramoorthy R,Fyfe PK,Hunter WN.Structure of Staphylococcus aureus Esx A suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein.J Mol Biol,2008,383(3):603-614.
[22]Burts ML,Williams WA,Debord K,et al.Esx A and Esx B are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections.Proc Natl Acad Sci USA,2005,102(4):1169-1174.
[23]Burts ML,Dedent AC,Missiakas DM.Esa C substrate for the ESAT-6 secretion pathway and its role in persistent infections of Staphylococcus aureus.Mol Microbiol,2008,69(3):736-746.
[24]Li H,Xu JJ,Chen W.The structure and function of M.tuberculosis RD-1 region encoded proteins.Prog Biochem Biophys,2009,36(10):1260-1266(in Chinese).李浩,徐俊杰,陈薇.结核分枝杆菌RD-1区编码蛋白的结构和功能.生物化学与生物物理进展,2009,36(10):1260-1266.
[25]Shi J,Zhu YK,Zheng DW,et al.Prokaryotic expression and immunogenicity analysis of mpt59 and esx A fusion gene from Mycobacterium tuberculosis.J Zhengzhou Univ:Med Sci,2015,50(6):761-765(in Chinese).石洁,朱岩昆,郑丹薇,等.结核分枝杆菌mpt59和esx A融合基因编码蛋白的原核表达及免疫原性观察.郑州大学学报,2015,50(6):761-765.
[26]Misstear K,Mc Neela EA,Murphy AG,et al.Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.J Infect Dis,2014,209(9):1479-1484.
[2]Yang YL,Yi SQ,Chen W.Vaccines and immunotherapeutic approaches against Staphylococcus aureus.Prog Microbiol Immunol,2012,40(2):47-52(in Chinese).杨益隆,易绍琼,陈薇.金黄色葡萄球菌疫苗及其免疫治疗进展.微生物学免疫学进展,2012,40(2):47-52.
[3]Chen YG,Yang DQ.Research progress of Staphylococcus aureus vaccine.Chin J Biol,2010,23(11):1271-1274(in Chinese).陈益国,阳大庆.金葡萄球菌疫苗的研究进展.中国生物制品学杂志,2010,23(11):1271-1274.
[4]Sundaramoorthy R,Fyfe PK,Hunter WN.Structure of Staphylococcus aureus Esx A suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein.J Mol Biol,2008,383(3):603-614.
[5]Otto M.Staphylococcus aureus toxins.Curr Opin Microbiol,2014,17:32-37.
[6]Anderson M,Aly KA,Chen YH,et al.Secretion of atypical protein substrates by the ESAT-6 secretion system of Staphylococcus aureus.Mol Microbiol,2013,90(4):734-743.
[7]Kneuper H,Cao ZP,Twomey KB,et al.Heterogeneity in ess transcriptional organization and variable contribution of the Ess/Type protein secretion systemⅦto virulence across closely Staphylococcus aureus strains.Mol Microbiol,2014,93(5):928-943.
[8]Zhou HQ,Du H,Zhang HF,et al.Esx A might as a virulence factor induce antibodies in patients with Staphylococcus aureus infection.Braz J Microbiol,2013,44(1):267-271.
[9]Bagnoli F,Fontana MR,Soldaini E,et al.Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus.Proc Natl Acad Sci USA,2015,112(12):3680-3685.
[10]Zhang BZ,Hua YH,Yu B,et al.Recombinant ESAT-6-like proteins provoke protective immune responses against invasive Staphylococcus aureus disease in a murine model.Infect Immun,2015,83(1):339-345.
[11]S?rensen AL,Nagai S,Houen G,et al.Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.Infect Immun,1995,63(5):1710-1717.
[12]Schukken YH,Leslie KE,Barnum DA,et al.Experimental Staphylococcus aureus intramammary challenge in late lactation dairy cows:quarter and cow effects determining the probability of infection.J Dairy Sci,1999,82(11):2393-2401.
[13]Stranger-Jones YK,Bae T,Schneewind O.Vaccine assembly from surface proteins of Staphylococcus aureus.Proc Natl Acad Sci USA,2006,103(45):16942-16947.
[14]Shao JG,Zhang BJ,Li SC,et al.Comparison and analysis of the biological of activity antiserum after immunizing with expressed different domains of Staphylococus aureus adhes in Fn BPA isolated from bovine milk.Chin J Animal Veter Sci,2015,46(7):1208-1214(in Chinese).邵俊高,张宝江李善春,等.牛乳源金黄色葡萄球菌黏附素分子Fn BPA不同功能区生物学活性的比较分析.畜牧兽医学报,2015,46(7):1208-1214.
[15]Jiang HJ,Su Y,Shen Y.Characterization of antiserum after co-immunized with adhesins expressing Clf A,Fn BPA-A and Fn BPA-BCD of Staphylococus aureus.Acta Microbiol Sin,2015,55(10):1343-1349(in Chinese).姜慧娇,苏艳,申煜.金黄色葡萄球菌黏附素Clf A,Fn BPA-A,Fn BPA-BCD联合免疫的免疫生物学特性.微生物学报,2015,55(10):1343-1349.
[16]Eric B,Pierre L,Lulzim S,et al.DNA immunization against the clumping factor A(Clf A)of Staphylococcus aureus.Vaccine,2002,20(17/18):2348-2357.
[17]Proctor RA.Challenges for a universal Staphylococcus aureus vaccine.Clin Infect Dis,2012,54(8):1179-1186.
[18]Kumar P,Chen K,Kolls JK.Th17 cell based vaccines in mucosal immunity.Curr Opin Immunol,2013,25(3):373-380.
[19]Korea CG,Balsamo G,Pezzicoli A,et al.Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells.Infect Immun,2014,82(10):4144-4153.
[20]Wang YN,Hu M,Liu Q,et al.Role of the ESAT-6secretion system in virulence of the emerging community-associated Staphylococcus aureus lineage ST398.Sci Rep,2016,6:25163.
[21]Sundaramoorthy R,Fyfe PK,Hunter WN.Structure of Staphylococcus aureus Esx A suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein.J Mol Biol,2008,383(3):603-614.
[22]Burts ML,Williams WA,Debord K,et al.Esx A and Esx B are secreted by an ESAT-6-like system that is required for the pathogenesis of Staphylococcus aureus infections.Proc Natl Acad Sci USA,2005,102(4):1169-1174.
[23]Burts ML,Dedent AC,Missiakas DM.Esa C substrate for the ESAT-6 secretion pathway and its role in persistent infections of Staphylococcus aureus.Mol Microbiol,2008,69(3):736-746.
[24]Li H,Xu JJ,Chen W.The structure and function of M.tuberculosis RD-1 region encoded proteins.Prog Biochem Biophys,2009,36(10):1260-1266(in Chinese).李浩,徐俊杰,陈薇.结核分枝杆菌RD-1区编码蛋白的结构和功能.生物化学与生物物理进展,2009,36(10):1260-1266.
[25]Shi J,Zhu YK,Zheng DW,et al.Prokaryotic expression and immunogenicity analysis of mpt59 and esx A fusion gene from Mycobacterium tuberculosis.J Zhengzhou Univ:Med Sci,2015,50(6):761-765(in Chinese).石洁,朱岩昆,郑丹薇,等.结核分枝杆菌mpt59和esx A融合基因编码蛋白的原核表达及免疫原性观察.郑州大学学报,2015,50(6):761-765.
[26]Misstear K,Mc Neela EA,Murphy AG,et al.Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.J Infect Dis,2014,209(9):1479-1484.