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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 evaluate the immunological efficacy of three recombinant proteins, Rpfd and Rpfd1, Rpfd2. Methods From November 2011 to February 2012, Rpfd (group A), Rpfd1 (group A1) and Rpfd2 (group A2) were respectively immunized with specific pathogens at 0, 2 and 4 weeks (SPF) BALB / c mice, 14 mice in each group. 14 mice of the same kind were immunized with BCG (group B) and saline (group C) respectively. At the fifth week, seven mice in each group were taken from the eyeball to take the blood, and the serum specific antibodies, serum IFN-γ and IL-2 levels were detected by ELISA. At the eighth week, the remaining seven mice in each group were treated with 1 × 105 CFU Mycobacterium tuberculosis standard strain H37Rv was used to infect mice. At the 9th week, the level of serum cytokines IFN-γ and IL-2 were detected. Results (1) antibody levels: ① Rpfd antigen coating. A2 group (1.470 ± 0.455) was higher than that of group B (t = 6.634, P <0.05); the A2 group (1.470 ± 0.455) , P <0.05). ② Rpfd1 antigen coating. (1.073 ± 0.304) in group A was significantly higher than that in group B (0.573 ± 0.004) (t = 5.276, P <0.05); A1 was 1.368 ± 0.171 in group B (t = 17.20 , P <0.05). The A2 group (2.766 ± 0.245) was higher than the B group (t = 31.643, P <0.05). ③ The Rpfd2 antigen was coated. (1.055 ± 0.202) in group A1 was significantly higher than that in group B (0.538 ± 0.100) (t = 8.009, P <0.05); A2 group (1.605 ± 0.544) was higher than group B (t = 8.192 , P <0.05). (2) IFN-γ levels: ① before infection. Group A stimulated IFN-γ production in mice [(553.47 ± 132.00) pg / ml] than in group B (385.28 ± 129.07 pg / ml) (t = 3.150, P <0.05) (492.41 ± 211.74) pg / ml] in group A1 was significantly higher than that in group B [(335.36 ± 207.72) pg / ml] (t = 2.874, ± 223.07) pg / ml] than in group B (t = 3.15, P <0.05). (3) IL-2 levels: ① before infection. The level of IL-2 in group A stimulated [(1490.05 ± 215.35) pg / ml] was higher than that in group B [(718.70 ± 269.29) pg / ml] 357.40) pg / ml] was higher than that of group B (t = 7.903, P <0.05); Group A stimulated IL-2 production in mice [(806.81 ± 306.39) pg / ml] higher than that in group B [(335.26 ± 176.81) pg / ml] 143.75) pg / ml] than in group B (t = 15.90, P <0.05). Conclusion The Rpfd, Rpfd1 and Rpfd2 proteins play a dual role in the initiation of host humoral immune function and enhancement of host cell immune function, which may be a new immunization strategy for the prevention and treatment of Mtb infection.