结核分枝杆菌MPT64-ESAT6融合蛋白在小鼠体内诱导的免疫应答及其保护力

Immune responses and protective efficacy induced in mice by Mycobacterium tuberculosis MPT64-ESAT6 fusion protein

目的:测定MPT64与ESAT6融合蛋白在小鼠体内诱导的免疫应答及保护力.方法:将MPT64与ESAT6的融合蛋白皮下免疫小鼠3次,每次间隔2wk,ELISA测定免疫小鼠血清特异性抗体的滴度.最后一次免疫完成后4wk,分离部分免疫小鼠脾淋巴细胞,MTT法检测淋巴细胞刺激指数,ELISA检测悬液中IFNγ水平.另一部分免疫的BALB/c小鼠经尾静脉感染MTB毒株H37Rv,4wk后计数脾脏细菌负荷数.结果:MPT64ESAT6融合蛋白免疫小鼠血清特异性抗体滴度1∶1500,免疫小鼠后淋巴细胞刺激增殖指数为2.23,而生理盐水免疫组只有0.88;脾淋巴细胞悬液中诱发的IFNγ为(4.28±0.27)μg/L,显著高于生理盐水免疫组[(0.48±0.17)μg/L,P<0.05],但不及BCG免疫组[(5.18±0.31)μg/L].与生理盐水免疫组(细菌负荷6.45±0.17)相比较,融合蛋白免疫的小鼠,对攻击感染后抗MTB在脾脏中增殖有显著作用,细菌负荷为5.04±0.11,但与BCG免疫组相比脾脏细菌负荷减少甚微(细菌负荷4.38±0.22).结论:MPT64与ESAT6融合蛋白可作为新型结核疫苗的候选组分.

AIM： To evaluate humoral and cell-mediated immune responses by MPT64 and ESAT6 fusion proteins and to test protective efficacy against Mycobacterium tuberculosis （MTB） challenge. METHODS： BALB/c mice were immunized 3 times at 2-week interval subcutaneously on the back with fusion protein MPT64-ESAT6. In the spleen lymphocytes of immunized mice stimulation index （SI） was measured by MTr method and the level of secreted IFN-γ/ was detected by ELISA. Some of vaccinated BALB/c mice by fusion proteins were intravenously infected with 1 × 10^5 CFU MTB strain H37Rv. Four weeks later, bacterial load in spleens was determined. RESULTS ： The titer of sera specific antibody in BALB/c mice immunized with fusion expression protein MPT64-ESAT6 was 1 ： 1500. The SI of lymphocytes in fusion protein immunized group was 2.23, significandy higher than that of saline immunized group （0.88）. The IFN-γ/level in culture supernatant of spleen lymphocytes from the mice immunized with fusion proteins were （4.28 ±0.27 ） μg/L, significantly higher than that of saline immunized group [ （0.48 ± 0.17） μg/L, P 〈 0.05 ], but lower than that of Bacillus Calmette Guerin （BCG） immunized group [（5. 18 ± 0. 31） μg/L]. Compared with saline immunized mice（ bacterial load was 6.45 ± 0.17）, dramatic reductions were observed for MTB replication in the spleen from BALB/c mice immunized with fusion proteins （ bacteria load was 5.04 ± 0. 11 ） following a subsequent challenge, but the protective efficacy in mice immunized with MPT64- ESAT6 was not as good as that of BCG immunized group （bacterial load was 4.38 ± 0.22）. CONCLUSION ： MPT64 and ESAT6 fusion proteins could be used as a novel component of the new TB vaccine.