日本血吸虫副肌球蛋白合成肽的免疫学鉴定

Immunological Identification of a Synthetic Peptide from the Paramyosin of Schistosoma japonicum

目的鉴定日本血吸虫副肌球蛋白合成肽Sj97-P22。方法27只雌性C57BL/6小鼠随机均分为合成肽Sj97-P22免疫组、无关肽免疫组和PBS免疫组,分别于尾基部皮下注射与完全福氏佐剂等体积充分混匀的合成肽Sj97-P22(100μg)乳化物、无关肽(100μg)乳化物和PBS乳化物,抗原免疫剂量为100μg/(只.次),共免疫2次,间隔1周。于免疫后7～10d分离各组小鼠脾单个核细胞,运用流式细胞技术三色标记法检测其CD4+T细胞胞内因子干扰素γ(IFN-γ)和白介素4(IL-4)。将Sj97-P22免疫小鼠或PBS免疫小鼠的脾单个核细胞分别与Sj97-P22、无关肽或PBS共培养,采用3H-TdR掺入法观察细胞增殖效果,并用ELISA法检测细胞培养上清中IL-2、IFN-γ和IL-4的浓度。结果Sj97-P22免疫组小鼠脾脏CD4+T细胞中胞内分泌IFN-γ的细胞百分率为(8.05±0.54)%,显著高于无关肽免疫组[(4.74±1.04)%]和PBS免疫组[(6.51±0.49)%](P<0.05);而分泌IL-4的细胞百分率[(0.60±0.11)%]显著低于PBS免疫组[(1.31±0.27)%](P<0.05),与无关肽免疫组[(0.84±0.08)%]间的差异则无统计学意义(P>0.05)。Sj97-P22可明显刺激Sj97-P22免疫小鼠脾单个核细胞增殖,增殖指数达到3.12±1.59,细胞培养上清中IL-2和IFN-γ浓度分别为(9.13±1.54)和(39.75±9.69)pg/ml,与无关肽和PBS刺激相比显著升高(P<0.05),而IL-4浓度在3个刺激物间的差异无统计学意义(P>0.05)。Sj97-P22不能刺激PBS免疫鼠脾单个核细胞增殖及培养上清中IL-2、IFN-γ及IL-4浓度变化。结论Sj97-P22可能是C57BL/6小鼠特异的Th1型表位。

Objective To identify the immunologic property of a synthetic peptide Sj97-P22 from paramyosin of Schistosoma japonicum （Sj97）. Methods Twenty-seven female C57BL/6 mice were divided into 3 groups each with 9 mice, Sj97-P22, control peptide and PBS groups, and each mouse was respectively immunized twice （seven days interval） with 100 μg of Sj97-P22, control peptide or PBS, emulsified with equal value of complete Freund＇s adjuvant. Seven to ten days after the second immunization, the mouse spleen mononuclear cells were isolated for three-color flow eytometery to detect intraeellular cytokines IFN-gamma and IL-4. Then the spleen mononuelear cells were co-cultured with Sj97-P22, control peptide or PBS respectively, and the incorporation rate of 3H-thymidine, as well as the levels of IL-2, IFN-gamma and IL-4 in the cultured cell supernatant, were measured. Results In CD4^＋ T cells, the percentage of IFN-gamma-producing cells in Sj97-P22 group [（8.05±0.54）%] was significantly higher than that of the control peptide group [（4.74±1.04）%] or PBS group [（6.51±0.49）%] （P〈0.05）, while the proportion of IL-4-producing cells was significantly lower in Sj97-P22 group [（0.60±0.11）%] than that in PBS group [（1.31±0.27）%] （P〈0.05）. Also, compared with control peptide or PBS stimulation, Sj97-P22 was able to effectively stimulate the proliferation with the stimulation index （3.12±1.59） and a higher secretion of IL-2 [（9.13±1.54） pg/ml] and IFN-gamma [（39.75±9.69） pg/ml] of spleen mononuclear cells in Sj97-P22-immunized mice （P〈0.05）. Both Sj97-P22 and control peptide were not effective stimulators to the spleen mononuclear cells from mice of PBS group. Conclusion It is highly possible that Sj97-P22 is a Th1-type epitope specific for C57BL/6 mice.