Hsp65与IL-2融合蛋白诱导小鼠细胞免疫应答及保护力

目的研究Hsp65与hIL-2的融合蛋白在小鼠体内诱导的免疫应答及保护力。方法在大肠杆菌中诱导表达Hsp65与hIL-2的融合蛋白,通过Ni-NTA亲合柱纯化后的蛋白经鉴定后,与佐剂DDA和MPL联合免疫小鼠,连续免疫3次,每次间隔2周,最后一次免疫结束后两周,分离5只小鼠脾淋巴细胞,测定淋巴细胞增殖指数,IFN-γ和IL-2水平,以及特异性淋巴细胞杀伤功能,其余5只免疫小鼠用于MTB毒株攻击实验。结果获得融合蛋白可分别与抗Hsp65和抗hIL-2的单抗发生特异性反应。融合蛋白免疫小鼠后,小鼠脾淋巴细胞被有效活化,诱导产生的-γIFN和IL-2的水平以及CTL杀伤功能均显著高于BCG和单纯Hsp65免疫组（P〈0.05）。融合蛋白免疫组可有效抵抗MTB毒株攻击,脾脏细菌数显著减少（4.36±0.48）,提供的保护力与BCG相当（4.30±0.53）。结论Hsp65与hIL-2的融合蛋白是一种有效的亚单位疫苗,可用于TB的预防。

Cell-Mediated Immune Responses Induced by Hsp65 and hIL-2 Fusion Protein against Mycobacterium Tuberculosis Infection and Its Protective Efficacy in Mice

Objective To study the immune responses and protective efficacy induced by heat shock protein 65（Hsp65） and human interleukin 2（hIL-2） fusion protein in mice against tuberculosis infection.Methods Hsp65 and IL-2 fusion protein was expressed in E.coli and purified by Ni-NTA affinity chromatography.The fusion protein was administered to mice in an adjuvant combination of dimethyl dioctadecylammonium bromide（DDA） and monophosphoryl lipid A（MPL）.The immunization repeated three times every two weeks.2 wks after the final immunization,5 mice were used for analysis of lymphocyte proliferation index,IL-2,IFN-γ assay and cytotoxic T lymphocyte（CTL） activity in each group.The other 5 were used for a MTB virulent strain attack experiment.Results The fusion protein Hsp65-hIL-2 was purified through Ni-NTA system.The Western blot assay showed that the recombinant protein could respond with the IL-2 mAb and Hsp65 mAb,respectively.The spleen lymphocytes of the fusion protein-immunized mice showed apparent proliferation in response to Hsp65 protein.The fusion protein elicited strong levels of γ-IFN and IL-2 in the lymphocytes culture supernatants,significantly higher than that of the BCG immunized group and single Hsp65 group（P〈0.05）.The spleen cells from mice immunized with fusion protein showed killing activity to the corresponding target cells.A dramatic reduction in the numbers of MTB colony forming units in the spleen was observed in the fusion protein group（4.36±0.48）,which provided equivalent protective effect to that afforded by BCG（4.30±0.53）.Conclusions Hsp65-IL-2 fusion protein is an effective subunit vaccine against tuberculosis.