表达结核分枝杆菌ESAT6-CFP10融合蛋白DNA疫苗免疫原性

Immunogenicity of DNA vaccine expressing ESAT6-CFP10 fusion protein of Mycobacterium tuberculosis

目的:研究表达结核分枝杆菌ESAT6-CFP10融合蛋白DNA疫苗在小鼠体内诱导的体液和细胞免疫应答以及对结核分枝杆菌(MTB)感染小鼠的保护能力.方法:以100μg重组质粒pcDNA-e6c10接种BALB/c小鼠腓前肌,共免疫3次.末次免疫结束2wk后,检测免疫小鼠特异性抗体滴度、淋巴细胞增殖指数、CTL杀伤效应以及诱导IFN-γ和IL-2水平.另一部分免疫的BALB/c小鼠以1×105MTB毒株H37Rv经尾静脉进行攻击,4wk后计数脾脏细菌负荷数,观察免疫小鼠对MTB抵抗作用.结果:表达ESAT6-CFP10融合蛋白DNA疫苗免疫小鼠血清特异性抗体滴度为1∶800.淋巴细胞刺激增殖指数为2.42±0.13,显著高于生理盐水对照组;免疫小鼠诱导IFN-γ含量(2449±12)ng/L与卡介苗(BCG)组无明显差异,IL-2含量(198±16)ng/L不及BCG免疫组,但显著高于生理盐水对照组;同时融合蛋白诱导的CTL杀伤率为42%.与生理盐水免疫组(细菌负荷6.51±0.13)相比较,DNA疫苗免疫的BALB/c小鼠对攻击感染后MTB在脾脏中增殖有较明显抵抗作用(细菌负荷4.51±0.23,P<0.05),但与BCG免疫组相比脾脏细菌负荷无明显减少.结论:表达ESAT6-CFP10融合蛋白DNA疫苗能在结核病预防中有一定免疫治疗作用.

AIM： To evaluate the humoral and cellular immune response induced by the DNA vaccine expressing ESAT6-CFP10 fusion protein and to test its protective efficacy against Mycobacterium tuberculosis （MTB） challenge. METHODS ： BALB/c mice were immunized intramuscularly three times with 100 μg recombinant plasmid pcDNA-e6e10. Two weeks after last immunization, the specific antibody titer and the stimulation index（SI） of spleen lymphocytes from the immunized mice were measured, and the levels of IFN-γ and IL-2 and the activity of antigen-specific CTL were detected. The DNA vaccine-vaccinated BALB/c mice were infected with 1 × 10^5 CFU （colony forming unit ） MTB H37Rv through tail vein. Four weeks later, the bacteria load in spleen was determined. RESULTS： The titer of serum specific antibody in BALB/c mice immunized with DNA vaccine was 1：800. The SI of DNA vaccine-immunized groups（ 2.42 ± 0.13 ） was significantly higher than that of saline-immunized group. The IFN-γ [ （2449 ± 12） ng/L] induced by DNA vaccine was not different from that in bacillus calmette guettn （ BCG ）-immunized group, while IL-2 [ （198 ± 16） ng/L] induced by DNA vaccine had significant difference from that of saline-immunized group and was lower than that of BCG-immunized group. The antigen-specific CTL efficacy was 42%. Compared with the saline immunized mice （bacteria load was 6.51 ±0.13）, a dramatic reduction of MTB replication was observed in the spleen （bacteria load was 4.51 ±0.23, P 〈 0.05 ） of BALB/c mice immunized with DNA vaccine following asubsequent challenge, but the protective efficacy of DNA vaccine was lower than that of BCG vaccine. CONCLUSION： DNA vaccine expressing ESAT6-CFP10 fusion protein has a immunotherapeutic effect to prevent tuberculosis.