Please wait a minute...

中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (12): 9-17    DOI: 10.13523/j.cb.20191202
研究报告     
重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫诱导Th1和Th17细胞免疫应答抵抗肺炎链球菌感染 *
孙思1,邱喻兰2,颜菊荣1,杨静1,吴光英1,王玲1,胥文春1,**()
1 重庆医科大学检验医学院 临床检验诊断学教育部重点实验室 重庆 400016
2 重庆医科大学附属儿童医院核医学实验室 重庆 400016
Recombinant Plasmid pcDNA3-dnaJ Prime/DnaJ Protein Boost Immunization Induce Th1/Th17 Immune Responses and Protect Mice Against Pneumococcal Infection
SUN Si1,QIU Yu-lan2,YAN Ju-rong1,YANG Jing1,WU Guang-ying1,WANG Lin1,XU Wen-chun1,**()
1 Key Laboratory of Diagnostics Medicine Designated by the Chinese Ministry of Education,Chongqing Medical University, Chongqing 400016, China
2 Laboratory of Nuclear Medicine, Children’s Hospital of Chongqing Medical University, Chongqing 400016, China;
 全文: PDF(1972 KB)   HTML
摘要：

目的 探索更有效的肺炎链球菌DNA疫苗和疫苗免疫策略,并探究其中的保护机制。方法 构建重组质粒pcDNA3-dnaJ并表达DnaJ蛋白,实验分别设置重组质粒pcDNA3-dnaJ/蛋白DnaJ免疫小鼠组及单独质粒pcDNA3-dnaJ免疫小鼠组,分别比较肺炎链球菌菌株攻毒后小鼠鼻腔灌洗液细菌载量及生存率,采用ELISA检测免疫小鼠血清抗体效价及炎症因子,流式细胞术分析体外BMDCs激活情况及Th1和Th17细胞免疫应答。结果 质粒pcDNA3-dnaJ免疫3次可诱导血清中抗原特异性抗体的产生,并减少肺炎链球菌攻毒后鼻咽部的细菌载量,但在防止致死性感染方面效果较差。然而,与重复质粒DNA接种三次相比,pcDNA3-dnaJ 1次/ DnaJ蛋白加强1次的免疫策略可以显著减少鼻咽中的肺炎链球菌定植,并能够更好的预防致死性感染。此外,与DNA质粒加强免疫相比,DnaJ蛋白加强免疫后可产生更高水平的IFN-γ和IL-17A。结论 重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫可能通过活化树突状细胞,进而诱导Th1和Th17细胞免疫应答,抵抗肺炎链球菌感染。
关键词： DNA疫苗;  肺炎链球菌;  异源免疫;  菌载量;  生存率    
Abstract:

Objective: To explore more effective S. pneumoniae DNA vaccines and vaccine immunization strategies and explore their protective mechanisms.Methods: The recombinant plasmid pcDNA3-dnaJ was constructed and DnaJ protein was expressed. The recombinant plasmid pcDNA3-dnaJ/protein DnaJ immunized group and the plasmid pcDNA3-dnaJ immunized group were separately set to compare the nasal lavage of mice treated with S. pneumoniae strain. The bacterial load and survival rate of the liquid, serum antibody titer and inflammatory factors after challenged were measured by ELISA. The activation of BMDCs and the immune responses of Th1 and Th17 cells were analyzed by flow cytometry.Methods: The plasmid pcDNA3-dnaJ immunization three times induced antigen-specific antibody in serum and reduced the bacterial loads in the nasopharynx after challenge with live S. pneumoniae, but it was less effective in protecting against a lethal infection. However, compared with repeating the plasmid DNA innoculation three times, the strategy of pcDNA3-dnaJ prime one time/DnaJ protein boost one time could significantly reduce the pneumococcal colonization in the nasopharynx and provid better protection against lethal infection. Furthermore, DnaJ protein boosting generated higher levels of IFN-γ and IL-17A than the DNA boosting.Conclusion: Compared with DNA plasmid booster, immunization using DNA prime/protein boost of pneumococcus protein may be a new strategy to develop vaccines against pneumococcal infection.
Key words: DNA vaccine    Streptococcus pneumonia    Heterologous immunity    Bacterial loads Lethal infection
收稿日期: 2019-04-19 出版日期: 2020-01-15
ZTFLH:  Q816  
基金资助: * 国家自然科学基金(81671639)
通讯作者: 胥文春     E-mail: xuwen@cqmu.edu.com
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
孙思
邱喻兰
颜菊荣
杨静
吴光英
王玲
胥文春

引用本文:

孙思,邱喻兰,颜菊荣,杨静,吴光英,王玲,胥文春. 重组质粒pcDNA3-dnaJ/蛋白DnaJ异源免疫诱导Th1和Th17细胞免疫应答抵抗肺炎链球菌感染 *[J]. 中国生物工程杂志, 2019, 39(12): 9-17.

SUN Si,QIU Yu-lan,YAN Ju-rong,YANG Jing,WU Guang-ying,WANG Lin,XU Wen-chun. Recombinant Plasmid pcDNA3-dnaJ Prime/DnaJ Protein Boost Immunization Induce Th1/Th17 Immune Responses and Protect Mice Against Pneumococcal Infection. China Biotechnology, 2019, 39(12): 9-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191202    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I12/9

图1  质粒pcDNA3-dnaJ构建及验证
图2  质粒pcDNA3-dnaJ诱导BMDCs的活化与成熟
图3  免疫小鼠后细菌载量和生存率
图4  免疫小鼠血清抗体效价
图5  免疫小鼠的细胞免疫应答
[1] O’Brien K L, Wolfson L J, Watt J P , et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet, 2009,374(9693):893-902
[2] WHO. Pneumococcal vaccines WHO position paper--2012. Releve Epidemiologique Hebdomadaire, 2012,87(14):129-144.
[3] Scott J A . The preventable burden of pneumococcal disease in the developing world. Vaccine, 2007,25(13):2398-2405.
[4] Wang Y, Yue X, Jin H , et al. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice. J Virol Methods, 2015,225:35-40.
[5] Li W, Wang S, Lu S . Pilot study on the use of DNA priming immunization to enhance Y. pestis LcrV-specific B cell responses elicited by a recombinant LcrV protein vaccine. Vaccines (Basel), 2013,2(1):36-48.
[6] Li P, Cao R B, Zheng Q S , et al. Enhancement of humoral and cellular immunity in mice against Japanese encephalitis virus using a DNA prime-protein boost vaccine strategy. Vet J, 2010,183(2):210-216.
[7] Golshani M, Rafati S, Siadat S D , et al. Improved immunogenicity and protective efficacy of a divalent DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA priming and protein boosting regimen. Mol Immunol, 2015,66(2):384-391.
[8] Wu K, Zhang X, Shi J , et al. Immunization with a combination of three pneumococcal proteins confers additive and broad protection against Streptococcus pneumoniae infections in mice. Infect Immun, 2010,78(3):1276-1283.
[9] Nguyen C T, Kim S Y, Kim M S , et al. Intranasal immunization with recombinant PspA fused with a flagellin enhances cross-protective immunity against Streptococcus pneumoniae infection in mice. Vaccine, 2011,29(34):5731-5739.
[10] Mann J F, Mckay P F, Arokiasamy S , et al. Pulmonary delivery of DNA vaccine constructs using deacylated PEI elicits immune responses and protects against viral challenge infection. J Control Release, 2013,170(3):452-459.
[11] Romani N, Gruner S, Brang D , et al. Proliferating dendritic cell progenitors in human blood. J Exp Med, 1994,180(1):83-93.
[12] Sallusto F, Lanzavecchia A . Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med, 1994,179(4):1109-1118.
[13] Kapsenberg M L . Dendritic-cell control of pathogen-driven T-cell polarization. Immunology, 2003,3(12):984-993.
[14] Banchereau J, Steinman R M . Dendritic cells and the control of immunity. Nature, 1998,392(6673):245-252.
[15] Kushwah R, Hu J . Complexity of dendritic cell subsets and their function in the host immune system. Immunology, 2011,133(4):409-419.
[16] Schraml B U, Reis E, Sousa C . Defining dendritic cells. Curr Opin Immunol, 2015,32:13-20.
[17] Guermonprez P, Valladeau J, Zitvogel L , et al. Antigen presentation and T cell stimulation by dendritic cells. Annual Review of Immunology, 2002,20(1):621-667.
[18] Jonuleit H, Kuhn U, Muller G , et al. Pro-inflammatory cytokines and prostaglandins induce maturation of potent immunostimulatory dendritic cells under fetal calf serum-free conditions. Eur J Immunol, 1997,27(12):3135-3142.
[19] Grant E V, Thomas M, Fortune J , et al. Enhancement of plasmid DNA immunogenicity with linear polyethylenimine. Eur J Immunol, 2012,42(11):2937-2948.
[20] Wegmann F, Gartlan K H, Harandi A M , et al. Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens. Nat Biotechnol, 2012,30(9):883-888.
[21] Mann J F, Mckay P F, Arokiasamy S , et al. Pulmonary delivery of DNA vaccine constructs using deacylated PEI elicits immune responses and protects against viral challenge infection. J Control Release, 2013,170(3):452-459.
[22] Lu Y J, Gross J, Bogaert D , et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog, 2008,4(9):e1000159.
[23] Zhang Z, Clarke T B, Weiser J N . Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. J Clin Invest, 2009,119(7):1899-1909.
[24] Wang W, Zhou A, Zhang X , et al. Interleukin 17A promotes pneumococcal clearance by recruiting neutrophils and inducing apoptosis through a p38 mitogen-activated protein kinase-dependent mechanism in acute otitis media. Infect Immun, 2014,82(6):2368-2377.
[25] Wilson R, Cohen J M, Jose R J , et al. Protection against Streptococcus pneumoniae lung infection after nasopharyngeal colonization requires both humoral and cellular immune responses. Mucosal Immunol, 2015,8(3):627-639.
[26] Blair C, Naclerio R M, Yu X , et al. Role of type 1 T helper cells in the resolution of acute Streptococcus pneumoniae sinusitis: a mouse model. J Infect Dis, 2005,192(7):1237-1244.
[27] Jahn-Schmid B, Messner P, Unger F M , et al. Toward selective elicitation of TH1-controlled vaccination responses: vaccine applications of bacterial surface layer proteins. Journal of Biotechnology, 1996,44(1-3):225-231.
[1] 程旭,杨雨睛,吴赛男,侯勤龙,李咏梅,韩慧明. 金黄色葡萄球菌SarA、IcaA及其融合基因的DNA疫苗构建及在小鼠免疫应答中的初步研究 *[J]. 中国生物工程杂志, 2020, 40(7): 41-50.
[2] 王猛,宋慧茹,程雨洁,王毅,杨波,胡征. 以核糖体蛋白L7/L12为分子标志物精准检测肺炎链球菌的研究 *[J]. 中国生物工程杂志, 2020, 40(4): 34-41.
[3] 黄健, 黄美容, 朱杰华, 骆诗露, 闵迅. 肺炎链球菌SP0306蛋白的表达纯化及结晶研究[J]. 中国生物工程杂志, 2015, 35(6): 21-25.
[4] 李楠, 王南杰, 王红翠, 李慧, 詹胜, 阳小燕, 孙雪松. 肺炎链球菌去信号肽脂蛋白PsaA锌离子结合特性的初步表征[J]. 中国生物工程杂志, 2011, 31(12): 27-32.
[5] 钟一维 李金耀 耿爽 王宾. 鉴定免疫细胞中DNA疫苗结合蛋白[J]. 中国生物工程杂志, 2010, 30(10): 0-0.
[6] 钟一维, 李金耀, 耿爽, 王宾. 鉴定免疫细胞中DNA疫苗结合蛋白[J]. 中国生物工程杂志, 2010, 30(10): 12-16.
[7] 张继文 杨桂连 王春凤. 逆转录病毒载体在基因工程疫苗方面的应用[J]. 中国生物工程杂志, 2010, 30(06): 130-133.
[8] 朱路 龙全科 刘海峰 鲁明波 胡媛 石佑恩 余龙江. 日本血吸虫三价DNA疫苗pVIVO2SjFABP/Sj26.SjGAPDH的构建及其免疫保护作用评价[J]. 中国生物工程杂志, 2010, 30(05): 36-42.
[9] 胡慧 陶玲 崔保安 黄弋 王汉中. 以减毒沙门氏菌为SARS-CoV N DNA口服疫苗载体的初步研究[J]. 中国生物工程杂志, 2010, 30(01): 41-46.
[10] 刘春国 刘明 李洪涛 杜金玲 张新涛 石薇霖. H1亚型猪流感病毒HA基因DNA疫苗的构建及其对Balb/c小鼠的免疫效果评价[J]. 中国生物工程杂志, 2009, 29(10): 38-43.
[11] 余云舟,李娜,王双,俞炜源,孙志伟. 电脉冲和布吡卡因增强A型肉毒毒素DNA核酸疫苗的免疫效果[J]. 中国生物工程杂志, 2009, 29(05): 33-38.
[12] 黄镜贤,曹以诚,杜正平,陶嫦立,杨化强. 共表达siRNA和hIL-12的新型乙肝多表位DNA疫苗的研究[J]. 中国生物工程杂志, 2008, 28(8): 36-42.
[13] 汪永信,张部昌,靳彦文,曹诚,钟辉,李平,张艳红,马清钧. pcD-awte候选疟疾DNA疫苗制备及其质量相关性分析[J]. 中国生物工程杂志, 2007, 27(4): 1-5.
[14] 杨化强 曹以诚. 丙型肝炎病毒多表位DNA疫苗的研究进展[J]. 中国生物工程杂志, 2007, 27(1): 119-125.
[15] 谭文杰,张柯,阮力. 丙型肝炎病毒（HCV）实验性疫苗的研究进展[J]. 中国生物工程杂志, 2006, 26(10): 62-68.
主管：中国科学院 　主办：中国科学院文献情报中心 　中国生物技术发展中心 　中国生物工程学会