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

Immune response induced by Micrococcus luteus Rpf domain and its mutants in mice

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。

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.