| | | 表达结核分枝杆菌ESAT6
| | 预防医学论文 作者：张海1，师长宏1，王丽梅2，薛莹2，柏银兰2，徐志凯
【关键词】 分枝杆菌,结核；疫苗,DNA；ESAT6；CFP10；融合蛋白；免疫原性
Immunogenicity of DNA vaccine expressing ESAT6CFP10 fusion protein of Mycobacterium tuberculosis
【Abstract】 AIM: To evaluate the humoral and cellular immune response induced by the DNA vaccine expressing ESAT6CFP10 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 pcDNAe6c10. 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 IFNγ and IL2 and the activity of antigenspecific CTL were detected. The DNA vaccinevaccinated BALB/c mice were infected with 1×105 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 vaccineimmunized groups(2.42±0.13) was significantly higher than that of salineimmunized group. The IFNγ［(2449±12) ng/L］induced by DNA vaccine was not different from that in bacillus calmette guerin(BCG)immunized group, while IL2［(198±16) ng/L］ induced by DNA vaccine had significant difference from that of salineimmunized group and was lower than that of BCGimmunized group. The antigenspecific 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 a subsequent challenge, but the protective efficacy of DNA vaccine was lower than that of BCG vaccine. CONCLUSION: DNA vaccine expressing ESAT6CFP10 fusion protein has a immunotherapeutic effect to prevent tuberculosis.
【Keywords】 Mycobacterium tuberculosis; vaccines, DNA; ESAT6; CFP10; fusion protein; immunogenicity
【摘要】 目的预防医学论文： 研究表达结核分枝杆菌ESAT6CFP10融合蛋白DNA疫苗在小鼠体内诱导的预防医学论文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100 μg重组质粒pcDNAe6c10接种BALB/c小鼠腓前肌，共免疫3次. 末次免疫结束2 wk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IFNγ和IL2水平. 另一部分免疫的BALB/c小鼠以1×105 MTB毒株H37Rv经尾静脉进行攻击，4 wk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ESAT6CFP10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IFNγ含量（2449±12）ng/L与卡介苗（BCG）组无明显差异，IL2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ESAT6CFP10融合蛋白DNA疫苗能在结核病预防中有一定免疫治疗作用.
【关键词】 分枝杆菌，结核；疫苗，DNA；ESAT6；CFP10；融合蛋白；免疫原性
0引言
研究认为卡介苗（bacillus calmette querin, BCG）可预防并减轻儿童的严重结核病（tuberculosis, TB），但对成人TB的预防作用从0到80％不等［1］. 因此急需研究一种比BCG更好的疫苗来控制该病的传播与蔓延. ESAT6抗原是结核分枝杆菌（Mycobacterium tuberculosis, MTB）早期分泌性低分子质量蛋白，能诱导机体产生强烈T细胞免疫应答和释放高水平IFNγ. 许多研究表明ESAT6蛋白具有良好的抗原刺激性并能被大多数TB患者所识别. 近年来又发现另一种低分子质量MTB培养滤液蛋白CFP10，CFP10与ESAT6同属于ESAT6家族，且由同一操纵子调控，与ESAT6相同的是，CFP10也是免疫优势抗原，能诱导机体产生强烈免疫应答［2］. 为了全面评估表达ESAT6CFP10融合蛋白DNA疫苗免疫学特性，我们研究了此种疫苗在小鼠体内诱导的体液和细胞免疫应答水平，同时测定免疫小鼠对MTB毒株H37Rv攻击的保护力，以了解其对TB的预防作用.
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