人结核分枝杆菌和牛结核分枝杆菌差异蛋白质组学的研究

目的 探讨人MTB标准株H37Rv与牛分枝杆菌标准株卡介苗株的亚细胞蛋白质组学差异.方法 应用密度梯度离心法分离H37Rv和卡介苗株的细胞壁、细胞膜和细胞质蛋白,利用二维液相色谱分离技术进一步获得H37Rv和卡介苗株亚细胞蛋白质组,采用酶联免疫吸附试验分别检测H37Rv和卡介苗各组分与结核病患者和健康人各5例血清的免疫学反应.组间比较采用t检验.结果 共获得26个特异性引发免疫反应的H37Rv菌株蛋白组分.细胞膜组分M3Fr18与结核病患者的血清免疫学反应的吸收光度(A450值)为(721±3)×10-3,明显高于健康人(356±6)×10-3],也明显高于卡介苗株细胞膜相应组分与健康人的血清免疫学反应强度(414±7)×10-3],差异均有统计学意义(t值分别为1.852和1.037,均P＜0.01).细胞膜组分MI0Fr21与结核病患者的血清免疫学反应的A450值为(954±6)×10-3,明显高于健康人(415±6)×10-3],也明显高于卡介苗株细胞膜相应组分与健康人的血清免疫学反应强度(315±4)×10-3],差异均有统计学意义(t值分别为2.113和2.550,均P＜0.01).结论 二维液相色谱和酶联免疫吸附试验联合应用,有助于检测人MTB感染的相关抗原蛋白.M3Fr18和M10Fr21蛋白质组分可引起结核病患者的特异性免疫反应,提示其可能是人MTB感染的特异抗原.

Abstract：

Objective To compare the proteome of Mycobacterium tuberculosis (MTB) H37 Rv strain with Bovine mycobacterium (Bacillus Calmette-Guerin, BCG) strain at the sub-cellular level. Methods Proteins of the cell wall, membrane and cytolymph of H37 Rv and BCG were extracted by density gradient centrifugation. Sub-cellular proteome of H37 Rv and BCG were analyzed using 2-dimensional liquid chromatography. The immunity reactions of H37 Rv fractions with sera from patients ( n = 5 ) and healthy controls ( n = 5 ), as well as BCG fractions with sera from healthy controls ( n = 5 ) were analyzed by ELISA.Data was analyzed by t test. Results Twenty-six fractions of H37 Rv were found to elicit specific antibody response. Fraction M3Fr18 of H37 Rv reacted with sera from patients. The A450 (721 ± 3 ) × 10-3] was higher than that with sera from healthy controls ( 356 ± 6) × 10 - 3 ] , as well as the A450 of the corresponding fractions of BCG with sera from healthy controls (414 ± 7) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 1. 852 and 1. 037, all P ＜ 0. 01 ). Moreover,fraction M10Fr21 of H37Rv reacted with sera from the patients. The A450 (954 ±6) × 10 -3 ] was higher than that with sera from the healthy controls (415 ± 6 ) × 10-3 ], as well as the A450 of the corresponding fractions of BCG with sera from the healthy controls ( 315 ± 4 ) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 2. 113 and 2. 550, all P ＜ 0. 01 ).Conclusions The combination of 2-dimensional liquid chromatography and immunology technology is useful in finding antigens associated with MTB infection. Fractions M3Fr18 and M10Fr21 can elicit specific immune reaction among MTB patients, suggesting that they may be specific antigens of MTB infection.

Comparison of the proteome of Mycobacterium tuberculosis with Bovine mycobacterium by immuno-proteomic technology

Objective To compare the proteome of Mycobacterium tuberculosis (MTB) H37 Rv strain with Bovine mycobacterium (Bacillus Calmette-Guerin, BCG) strain at the sub-cellular level. Methods Proteins of the cell wall, membrane and cytolymph of H37 Rv and BCG were extracted by density gradient centrifugation. Sub-cellular proteome of H37 Rv and BCG were analyzed using 2-dimensional liquid chromatography. The immunity reactions of H37 Rv fractions with sera from patients ( n = 5 ) and healthy controls ( n = 5 ), as well as BCG fractions with sera from healthy controls ( n = 5 ) were analyzed by ELISA.Data was analyzed by t test. Results Twenty-six fractions of H37 Rv were found to elicit specific antibody response. Fraction M3Fr18 of H37 Rv reacted with sera from patients. The A450 (721 ± 3 ) × 10-3] was higher than that with sera from healthy controls ( 356 ± 6) × 10 - 3 ] , as well as the A450 of the corresponding fractions of BCG with sera from healthy controls (414 ± 7) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 1. 852 and 1. 037, all P ＜ 0. 01 ). Moreover,fraction M10Fr21 of H37Rv reacted with sera from the patients. The A450 (954 ±6) × 10 -3 ] was higher than that with sera from the healthy controls (415 ± 6 ) × 10-3 ], as well as the A450 of the corresponding fractions of BCG with sera from the healthy controls ( 315 ± 4 ) × 10-3 ]. The differences between the patient group and the 2 healthy control groups were significant ( t = 2. 113 and 2. 550, all P ＜ 0. 01 ).Conclusions The combination of 2-dimensional liquid chromatography and immunology technology is useful in finding antigens associated with MTB infection. Fractions M3Fr18 and M10Fr21 can elicit specific immune reaction among MTB patients, suggesting that they may be specific antigens of MTB infection.