A族链球菌多价疫苗的完善制备及其动物免疫保护作用研究
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摘要
目的:完善A族链球菌(GAS)多价疫苗并探究其免疫保护效果。方法:构建GAS的多肽疫苗F7M6,将其中的M6多肽克隆作为对照,表达纯化后,将两种蛋白连同本室保存的GAS M1蛋白分别免疫小鼠,末次免疫后10 d,半数小鼠行ELISPOT检测IL-4和IFN-γ,ELISA检测各组的Ig G、Ig G1、Ig G2a;同时,以F7M6蛋白为诊断抗原检测其与抗链"O"阳性人血清的特异性结合;致死量的M1GAS攻毒并计算存活率。结果:无论是诱导细胞免疫,还是诱导抗体产生,F7M6组的水平均为最高;攻毒后F7M6蛋白的生存率为66.7%。F7M6蛋白检测抗链"O"阳性血清的阳性检出率为95%。结论:GAS的多肽疫苗F7M6能诱导特异性细胞和体液免疫应答,对GAS感染具有良好的保护效果,且F7M6的多价表位涵盖于时下流行的大部分GAS的血清型中。
Objective: To optimize group A streptococcus( GAS) polyvalent epitope-based vaccine,and explore its immune protection effect. Methods: Psolyvalent vaccine of GAS,F7M6,was constructed and expressed,in which the M6 peptide was constructed as control. 12 mice of each group were immunized with 20 μg per dose of purified F7M6,M6 or M protein( stored in our lab),and PBS as negative control. Half of the mice in each group were sacrificed on 10 days of the last immunization,and the levels of Ig G,Ig G1 and Ig G2 a,were detected by ELISA and the levels of IL-4 and IFN-γ were detected by ELISPOT. At the same time,the left immunized animals were challenged with a lethal dose of M1 GAS,and determined the protective effect of the vaccine. Besides,the specific binding ability of F7M6 protein with the sera from ASO-positive patients was detected by ELISA. Results: In F7M6 group,the levels of not only sera Ig G,Ig G1,Ig G2 a,but also IL-4 and IFN-γ were the highest one compared with other groups. Following challenge with type M1 GAS,the survival rates of F7M6 group was 66. 7%. Additionally,when F7M6 acted as diagnostic antigen,the positive detective rate of ASO-positive patient sera was 95%. Conclusion: Polypeptides F7M6,could elicit the best humoral and cell-mediated immune response than other groups,and induce a protective effect in immunized mice against M1 GAS infection. Excitedly,the epitopes of F7M6 protein covers the most of GAS serotypes at present.
Objective: To optimize group A streptococcus( GAS) polyvalent epitope-based vaccine,and explore its immune protection effect. Methods: Psolyvalent vaccine of GAS,F7M6,was constructed and expressed,in which the M6 peptide was constructed as control. 12 mice of each group were immunized with 20 μg per dose of purified F7M6,M6 or M protein( stored in our lab),and PBS as negative control. Half of the mice in each group were sacrificed on 10 days of the last immunization,and the levels of Ig G,Ig G1 and Ig G2 a,were detected by ELISA and the levels of IL-4 and IFN-γ were detected by ELISPOT. At the same time,the left immunized animals were challenged with a lethal dose of M1 GAS,and determined the protective effect of the vaccine. Besides,the specific binding ability of F7M6 protein with the sera from ASO-positive patients was detected by ELISA. Results: In F7M6 group,the levels of not only sera Ig G,Ig G1,Ig G2 a,but also IL-4 and IFN-γ were the highest one compared with other groups. Following challenge with type M1 GAS,the survival rates of F7M6 group was 66. 7%. Additionally,when F7M6 acted as diagnostic antigen,the positive detective rate of ASO-positive patient sera was 95%. Conclusion: Polypeptides F7M6,could elicit the best humoral and cell-mediated immune response than other groups,and induce a protective effect in immunized mice against M1 GAS infection. Excitedly,the epitopes of F7M6 protein covers the most of GAS serotypes at present.
引文
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[22]O'Hagan DT,Ott GS,Gregorio ED,et al.The mechanism of action of MF59-an innately attractive adjuvant formulation[J].Vaccine,2012,30(29):4341-4348.
[2]Pichichero ME,Casey JR.Systematic review of factors contributing to penicillin treatment failure in Streptococcus pyogenes pharyngitis[J].Arch Otolaryngol Head Neck Surg,2008,137(6):851-857.
[3]Lucilla B,Roberta C,Simona R,et al.Therapeutic failures of antibiotics used to treat macrolide-susceptible Streptococcus pyogenes infections may be due to biofilm formation[J].J Clin Microbiol,2006,44(8):2721-2727.
[4]Ma C,Zheng L,Li W,et al.Fba A-and M protein-based multiepitope vaccine elicits strong protective immune responses against group A streptococcus in mouse model[J].Microbes Infect,2014,16(5):409-418.
[5]郭奕阳,马翠卿,魏澎,等.A族链球菌(GAS)Fba蛋白单克隆抗体对应表位核心氨基酸的确定[J].中国免疫学杂志,2009,25(12):1059-1062.
[6]马翠卿,顾海燕,郭奕阳,等.A族链球菌表面蛋白Fba单克隆抗体的制备及其对应表位的初步鉴定[J].中国免疫学杂志,2008,24(11):1046-1048.
[7]魏澎,马翠卿,郭奕阳,等.A族链球菌表面蛋白Fba保护性区段的鉴定[J].中华传染病杂志,2010,28(5):257-261.
[8]梁云梅,常贺生,沈叙庄,等.北京地区儿童携带的酿脓链球菌emm分型和超抗原spe A及spe C的相关性研究[J].中华实用儿科临床杂志,2010,25(22):1700-1702.
[9]梁云梅,常贺生,沈叙庄,等.致儿童咽扁桃体炎酿脓链球菌emm分型[J].临床儿科杂志,2011,29(4):382-385.
[10]Terao Y,Kawabata S,Kunitomo E,et al.Fba,a novel fibronectinbinding protein from Streptococcus pyogenes,promotes bacterial entry into epithelial cells,and the fba gene is positively transcribed under the Mga regulator[J].Mol Microbiol,2001,42(1):75-86.
[11]Cui QM,Cai HL,Xiu RW,et al.Similar ability of Fba A with M protein to elicit protective immunity against group A streptococcus challenge in mice[J].Cell Mol Immunol,2009,6(1):73-77.
[12]Goodman RE.Practical and predictive bioinformatics methods for the identification of potentially cross-reactive protein matches[J].Mol Nutr Food Res,2006,50(7):655-660.
[13]Courtney HS,Hasty DL,Dale JB.Serum opacity factor(SOF)of Streptococcus pyogenes evokes antibodies that opsonize homologous and heterologous SOF-positive serotypes of group A streptococci[J].Infect Immun,2003,71(9):5097-5103.
[14]Mcarthur J,Medina E,Mueller A,et al.Intranasal vaccination with streptococcal fibronectin binding protein Sfb1 fails to prevent growth and dissemination of Streptococcus pyogenes in a murine skin infection model[J].Infect Immun,2004,72(12):7342-7345.
[15]Samanidou VF,Nika MK,Papadoyannis IN.HPLC as a tool in medicinal chemistry for the monitoring of tricyclic antidepressants in biofluids[J].Mini Rev Med Chem,2008,8(3):256-275.
[16]Ramachandran V,Mcarthur JD,Behm CE,et al.Two distinct genotypes of prt F2,encoding a fibronectin binding protein,and evolution of the gene family in streptococcus pyogenes[J].J Bacteriol,2004,186(22):7601-7609.
[17]Cue D,Dombek PE,Lam H,et al.Streptococcus pyogenes serotype M1 encodes multiple pathways for entry into human epithelial cells[J].Infect Immun,1998,66(10):4593-4601.
[18]李彩虹,马翠卿,王锦,等.A族链球菌表面新发现蛋白Fba真核表达质粒的构建及其诱导的免疫应答[J].中国免疫学杂志,2007,23(9):835-838.
[19]马翠卿,李彩虹,王秀荣,等.A族链球菌表面蛋白Fba的原核表达及免疫原性分析[J].中华传染病杂志,2008,26(3):146-150.
[20]Traquina P,Morandi M,Contorni M,et al.MF59 adjuvant enhances the antibody response to recombinant hepatitis B surface antigen vaccine in primates[J].J Infect Dis,1996,174(6):1168-1175.
[21]Granoff DM,Mchugh YE,Raff HV,et al.MF59 adjuvant enhances antibody responses of infant baboons immunized with haemophilus influenzae Type b and Neisseria meningitidis group C oligosaccharide-CRM197 conjugate vaccine[J].Infect Immun,1997,65(5):1710-1715.
[22]O'Hagan DT,Ott GS,Gregorio ED,et al.The mechanism of action of MF59-an innately attractive adjuvant formulation[J].Vaccine,2012,30(29):4341-4348.