结核亚单位疫苗AEC/BC-C02诱导小鼠长期的抗原特异性细胞应答  

M y cobacterium tuberculosis subunit vaccine AEC/BC-C02induced long term antigen-specific cellular immune response in mice

作　　者：卢锦标[1] 杨蕾[1] 付丽丽[2] 陈保文[1] 都伟欣[1] 王国治[1] 徐苗[1] 

机构地区：[1]中国食品药品检定研究院结核病疫苗室,北京100050 [2]温州医学院环境与公共卫生学院

出　　处：《中国防痨杂志》2013年第1期32-36,共5页Chinese Journal of Antituberculosis

基　　金："十二五"国家科技重大专项(2012ZX10004701)

摘　　要：目的动态评价结核亚单位候选疫苗AEC/BC-C02在小鼠中的细胞免疫应答水平。方法6～8周龄SPF级BALB/c雌鼠48只,按数字表法随机分成两组,一组免疫AEC/BC-C02,另一组免疫PBS。末次免疫后第1、2、4、8周分别取两组小鼠(6只/组)分离脾淋巴细胞,ELISPOT检测分泌抗原特异性IFN-γ的T细胞频率;在第2、4、8周ELISA检测抗原特异性IFN-γ的分泌量;在第4、8周,Cell Counting Kit-8(CCK-8)法检测脾淋巴细胞增殖。结果 (1)末次免疫后第1、2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B特异性的IFN-γ斑点形成细胞(spot forming cells,SFC)分别为168.8±103.5、205.2±51.0、206.8±65.3和160.0±67.9,与PBS对照组的8.9±6.0、16.1±18.8、9.3±4.9和7.7±6.6比较,差异均有统计学意义(成组t检验,t值分别为3.779、8.525、7.424、5.473;P值均<0.01);EC特异性的IFN-γSFC分别为45.1±18.6、75.6±39.3、86.2±50.4和54.3±26.3,与PBS对照组的4.5±3.5、11.7±10.5、3.8±5.8和5.2±4.3比较,差异均有统计学意义(成组t检验,t值分别为5.258、3.850、3.977、4.521;P值均<0.01)。(2)末次免疫后第2、4、8周,对疫苗组小鼠脾淋巴细胞,Ag85B多肽刺激的IFN-γ分泌量分别是(1.27±0.13)ng/ml,(1.76±0.55)ng/ml和(1.44±0.44)ng/ml;EC多肽刺激的IFN-γ分泌量分别是(0.81±0.33)ng/ml,(0.81±0.69)ng/ml和(0.54±0.29)ng/ml,两者间差异有统计学意义(配对t检验,分别为:t=3.008,P<0.05;t=2.631,P<0.05;t=10.02,P<0.01)。(3)末次免疫后第4、8周,Ag85B多肽对疫苗组小鼠脾淋巴细胞的刺激指数(SI)分别为1.756±0.339和1.936±0.287,均分别高于PBS组的1.287±0.0581和1.382±0.114,差异有统计学意义(成组t检验,分别为:t=3.030,P<0.05;t=4.387,P<0.01);EC多肽对疫苗组SI分别为1.599±0.154和1.581±0.156,均分别高于PBS组的1.380±0.126和1.314±0.170,差异有统计学意义(成组t检验,t值分别为2.540、2.844;P值均<0.05)。结论新型结核亚单位疫苗AEC/BC-C02免疫小鼠可诱导长期稳定存在的抗原特异�Objective To evaluate dynamically cellular immune response of Mycobacterium tuberculosis subunit vaccine AEC/BC-C02 in mice. Methods Forty eight female BALB/c mice （6-8 weeks old） were randomly divided into two groups. One group was immunized intramuscularly 3 times with AEC/BC-C02 at an interval of 10 days, and the other with PBS as the control. At 1, 2, 4 and 8 weeks after the last vaccination, spleen lymphocytes were isolated （6 mice per group） to measure the frequency of antigen-specific IFN-γ secreting T cell by ELISPOT. Meanwhile, antigen-specific IFN-γ level was detected by ELISA at 2, 4 and 8 weeks, and lymphoproliferation response was measured by cell counting kit-8 assay at 4 and 8 weeks. Results （1） At 1, 2, 4 and 8 weeks after the last vaccination, the spot forming cells （SFC） of Ag85B-specific IFN-γ in vaccine group were respectively 168.8 ±103.5, 205.24±51.0, 206.84±65.3 and 160.04±67.9, and significantly higher than that in PBS group （8.94±6.0, t=3. 779, P〈0. 01; 16.1±18.8, t=8. 525, P〈0.01; 9.3±4.9, t=7. 424, P〈0.01 and 7.74±6.6, t=5. 473,P〈0.01, respectively）. ESAT6/CFP10 （EC）-specific IFN-γ SFC in vaccine group were respectively 45.1±18.6, 75.6±39.3, 86.2±50.4 and 54. 3±26.3, and significantly higher than that in PBS group（4. 5±3.5, t=5. 258,P〈0.01; 11.7±10.5, t=3. 850, P〈0.01; 3.8±5.8, t=3.977, P〈0.01 and 5.2±4.3, t=4.521, P〈0.01, respectively）. （2） At 2, 4 and 8 weeks after the last vaccination, IFNγ release induced by Ag85B peptide pool in vaccine group were （1. 274±0. 13） ng/ml, （1.76±0.55） ng/ml and （1.44±0. 44） ng/ml, respectively, and that in- duced by EC peptide pool were （0. 81±0.33） ng/ml, （0.81±0.69） ng/ml and （0.54±0.29） ng/ml, respectively.The difference between them was statistically significant （t=3. 008, P〈0.05; t= 2. 631, P〈0. 05 and t= 10. 02, P〈0.01, respectively）. （3） At 4 and 8 weeks after the last vaccination, stimulation indexes （SI） of spleen lymphocytes in

关 键 词：疫苗 亚单位 免疫 细胞 抗原 细菌 细菌蛋白质类 重组融合蛋白质类 小鼠 

分 类 号：R392[医药卫生—免疫学]