复苏促生长因子结构域及其突变体重组蛋白诱导小鼠免疫应答的研究

摘要/Abstract

摘要： 目的评价复苏促生长因子结构域蛋白（Rpfd）及其突变体蛋白（Rpfd1、Rpfd2）等3种重组蛋白的免疫效能。方法 2011年11月至2012年2月间，分别以本实验前期制备的重组蛋白Rpfd（A组）、Rpfd1（A1组）、Rpfd2（A2组）分别于0、2、4周免疫无特定病原体（SPF）级BALB/c小鼠，每组14只，并分别以BCG（B组）和生理盐水（C组）同期免疫同种小鼠各14只作为对照。第5周，每组取7只小鼠摘眼球取血，ELISA法检测血清特异性抗体、血清IFN-γ和IL-2表达水平；第8周，每组剩余7只小鼠用1×10⁵ CFU结核分枝杆菌标准株H37Rv感染小鼠，第9周检测感染后小鼠血清细胞因子IFN-γ和IL-2水平。结果（1）抗体水平：①Rpfd抗原包被。A1组刺激小鼠产生抗体A₄₅₀的检测值（0.990±0.272）高于B组（0.631±0.180）（t=4.635，P<0.05）；A2组（1.470±0.455）高于B组（t=6.634，P<0.05）。②Rpfd1抗原包被。A组刺激小鼠产生抗体A₄₅₀的检测值（1.030±0.304）高于B组（0.573±0.004）（t=5.276，P<0.05）；A1组（1.368±0.171）高于B组（t=17.20，P<0.05）；A2组（2.766±0.245）高于B组（t=31.643，P<0.05）；③Rpfd2抗原包被。A1组刺激小鼠产生抗体A₄₅₀的检测值（1.055±0.202）高于B组（0.538±0.100）（t=8.009，P<0.05）；A2组（1.605±0.544）高于B组（t=8.192，P<0.05）。（2） IFN-γ水平：①感染前。A组刺激小鼠产生IFN-γ水平［（553.47±132.00）pg/ml］高于B组［（385.28±129.07）pg/ml］（t=3.150，P<0.05）；②感染后。A1蛋白组刺激小鼠产生IFN-γ水平［（492.41±211.74）pg/ml］高于B组［（335.36±207.72）pg/ml］（t=2.874，P<0.05）；A2组［（543.09±223.07）pg/ml］高于B组（t=3.15，P<0.05）。（3）IL-2水平：①感染前。A组刺激小鼠产生IL-2水平［（1490.05±215.35）pg/ml］高于B组［（718.70±269.29）pg/ml］（t=7.763，P<0.05）；A1组［（1738.91±358.40）pg/ml］高于B组（t=7.903，P<0.05）；A2组［（2270.74±193.40）pg/ml］高于B组（t=16.308，P<0.05）；②感染后。A组刺激小鼠产生IL-2水平［（806.81±306.39）pg/ml］高于B组［（335.26±176.81）pg/ml］（t=4.627，P<0.05）；A1组［（1373.22±143.75）pg/ml］高于B组（t=15.90，P<0.05）。结论 Rpfd、Rpfd1、Rpfd2蛋白具有启动宿主体液免疫功能及增强宿主细胞免疫功能的双重作用，可能成为预防及治疗Mtb感染的免疫新策略。

关键词: 细菌蛋白质类, 白细胞介素2, 干扰素Ⅱ型, 重组蛋白质类, 抗体生成, 免疫,细胞

Abstract: Objective To study the immunogenicity of three recombinant proteins (Rpfd, Rpfd1, Rpfd2) of Micrococcus luteus resuscitation-promoting factor (Rpf) domain and its mutants in mice. Methods Seventy male-BALB/c mice were divided randomly into 5 groups as follow: Rpfd（A）, Rpfd1（A1）, Rpfd2（A2）, BCG（B）and Saline（C）．The mice were immunized subcutaneously at weeks 0, 2 and 4. At 1 week after the final immunization, 7 mice each group were collected and separated sera to detect levels of IFN-γ, IL-2 and antibodies against Rpfd, Rpfd1, Rpfd2 proteins in sera by ELISA. At 4 weeks after the final immunization, the other 7 mice were infected with 1×10⁵CFU Mycobacterium tuberculosis H37Rv by the tail vein injection. At 1 week after infection, the levels of IFN-γ and IL-2 were detected by ELISA. Results （1）Antibody levels: ①Rpfd specific antibody levels in mice：group A1（0.990±0.272）were higher than group B（0.631±0.180; t=4.635, P<0.05）; group A2（1.470±0.455）were higher than group B（ t=6.634, P<0.05）. ②Rpfd1 specific antibody levels：group A （1.030±0.304）were higher than group B（0.573±0.004; t=5.276, P<0.05）; group A1（1.368±0.171）were higher than group B（t=17.20, P<0.05）; group A2 （2.766±0.245）were higher than group B（t=31.643, P<0.05）. ③Rpfd2 specific antibody levels：group A1（1.055±0.202）were higher than group B（0.538±0.100; t=8.009, P<0.05）; group A2（1.605±0.544）were higher than group B（t=8.192,P<0.05）. （2）Levels of IFN-γ: Before infection, IFN-γ levels in mice of group A（553.47±132.00) pg/ml were higher than that of group B（385.28±129.07) pg/ml（t=3.150,P<0.05）.After infection, IFN-γ levels in mice of group A1（492.41±211.74）pg/ml were higher than that of group B（335.36±207.72) pg/ml（ t=2.874, P<0.05）; group A2（543.09±223.07）pg/ml were higher than group B（t=3.15, P<0.05）. （3） Levels of IL-2: Before infection, IL-2 levels in mice of group A（1490.05±215.35) pg/ml were higher than that of group B（718.70±269.29) pg/ml（t=7.763,P<0.05）; group A1（1738.91±358.40） pg/ml were higher than group B（t=7.903, P<0.05）; group A2（2270.74±193.40) pg/ml were higher than that of group B（t=16.308,P<0.05）. After infection, IL-2 levels in mice of group A（806.81±306.39) pg/ml were higher than that of group B（335.26±176.81） pg/ml（t=4.627, P<0.05）; group A1（1373.22±143.75） pg/ml were higher than that of group B（t=15.90, P<0.05）. Conclusion Vaccination with Rpfd、Rpfd1 and Rpfd2 proteins could induce humoral and cellular immune responses in BALB/c mice. They could be used as candidate vaccines.

Key words: Bacterial proteins, Interleukin-2, Interferon type Ⅱ, Recombinant proteins, Antibody formation, Immunity,cellular

邱奕赵善民师长宏史皆然. 复苏促生长因子结构域及其突变体重组蛋白诱导小鼠免疫应答的研究[J]. 中国防痨杂志, 2013, 35(5): 331-336.

QIU Yi, ZHAO Shan-min, SHI Chang-hong, SHI Jie-ran. Immune response induced by Micrococcus luteus Rpf domain and its mutants in mice[J]. Chinese Journal of Antituberculosis, 2013, 35(5): 331-336.

参考文献

［1］Derrick SC, Perera LP, Dheenadhayalan V, et al. The safety of post-exposure vaccination of mice infected with Mycobacterium tuberculosis. Vaccine, 2008, 26（48）: 6092-6098.
［2］吴雪琼. 新型结核病疫苗的研究现状与发展趋势.中国防痨杂志，2012，34 （3）：133-137.
［3］Kondratieva T, Rubakova E, Kana BD, et al. Mycobacterium tuberculosis attenuated by multiple deletions of Rpf genes effectively protects mice against TB infection. Tuberculosis(Edinb), 2011, 91（3）:219-223.
［4］Cohen-Gonsaud M, Keep NH, Davies AP, et al. Resuscitation-promoting factors possess a lysozyme-like domain. Trends Biochem Sci, 2004, 29（1）: 7-10.
［5］苏明权，岳乔红，杨柳，等. 5种结核分枝杆菌Rpf样蛋白的活性鉴定及促生长作用.中国人兽共患病学报，2008，24（9）: 841-845.
［6］岳晨莉，史皆然，师长宏，等. 藤黄微球菌复苏促进因子结构域及其突变体基因的克隆和表达及生物活性的测定.中华结核和呼吸杂志，2008，31（10）:3761-3765.
［7］Kana BD, Gordhan BG, Downing KJ, et al. The resuscitation-promoting factors of Mycobacterium tuberculosis are required for virulence and resuscitation from dormancy but are collectively dispensable for growth in vitro. Mol Microbiol, 2008, 67（3）:672-684.
［8］Fan A, Jian W, Shi C, et al. Production and characterization of monoclonal antibody against Mycobacterium tuberculosis RpfB domain. Hybridoma, 2010, 29（4）: 327-332.
［9］樊爱琳．Rpf蛋白结构域的生物学及免疫学特性的初步研究．西安：第四军医大学，2008．
［10］Dietrich J.Doherty TM. Interaction of Mycobacterium tuberculosis with the host: consequences for vaccine development. APMIS, 2009,117(5-6):440-457.
［11］黎友伦,罗永艾，王国治. 细胞因子及其受体在结核免疫中的作用. 国外医学内科学分册，2005，32（4）: 146-167.
［12］Flynn JL. Immunology of tuberculosis and implications in vaccine development. Tuberculosis（Edinb）, 2004,84(1-2): 93-101.
［13］Johnson BJ, Ress SR, Willcox P, et al. Clinical and immune responses of tuberculosis patients treated with low-dose IL-2 and multidrug therapy. Cytokines Mol Ther, 1995, 1（3）: 185-196.
［14］汤红明，刘君炎，高立芬． IFN-γ 与TNF 联合治疗结核菌感染小鼠疗效及机理的研究．免疫学杂志，2001，17（6）: 457-459．
［15］要玉霞，刘传玉．干扰素辅助治疗耐多药结核病的疗效观察．中国防痨杂志，2003，25（1）: 43-44．
［16］雷建平．重新审视结核病免疫治疗研究的方向和方法．中华临床医师杂志（电子版），2010，4（7）: 908-915．

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