结核杆菌热休克蛋白质70作为乙型肝炎核心抗原细胞毒性T淋巴细胞表位肽载体的研究

目的探讨结核杆菌热休克蛋白质70（TB．HSP70）作为乙型肝炎病毒（HBV）核心抗原细胞毒性T淋巴细胞（CTL）表位肽载体，诱导HBV特异性免疫应答的可能性。方法体外观察重组TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物诱导慢性乙型肝炎患者外周血淋巴细胞增殖以及HBV特异性细胞毒活性；以Balb／c小鼠为体内研究对象，行流式细胞术分析免疫后小鼠外周血和脾细胞中CD4^＋与CD8^＋T淋巴细胞及自然杀伤（NK）细胞的比率，并观察能否诱导HBV特异性免疫保护作用。结果体内外研究表明TB．HSP70-CTL融合蛋白质和TB．HSP70／CTL复合物能有效地诱导HBV特异性细胞毒活性，体内能激活CD4^＋与CD8^＋T淋巴细胞及NK细胞增殖。在体内，TB．HSP70-CTL融合蛋白质较复合物能更有效地激活免疫应答，其杀伤率为28．9％。CD8^＋T淋巴细胞在脾细胞中比率为43．9％，NK细胞为13．6％。而单纯的TB．HSP70和CTL表位肽并不能有效地引起机体的免疫应答。结论TB．HSP70能够作为乙型肝炎核心抗原CTL表位肽的载体，提高小分子表位肽的免疫原性。

Immune adjuvant effect of TB.HSP70 to its accompanying cytotoxic T lymphocytes epitope elicits HBV specific immune response

Objective To investigate whether Mycobacterium tuberculosis heat shock protein 70 (TB.HSP70) can be used as an adjuvant carrier to stimulate immune response to an accompanying cytotoxic T lymphocytes (CTL) epitope peptide from hepatitis B virus (HBV) core antigen. Methods In vitro, peripheral blood mononuclear cells (PBMCs) from chronic hepatitis B volunteers were stimulated with TB.HSP70-CTL fusion protein and TB.HSP70/CTL complex, and then HBV specific CTL activity was assessed. In vivo, the CD4+ T, CD8+T and natural killer cells (NKs) in the peripheral blood and in spleens of the immunized mice were measured by flow cytometry and the protective HBV specific immune responses of the mice were also evaluated. Results The results revealed that both of them could induce HBV specific CTL response in human PBMCs and in the immunized mice. In the mice they activated CD4+T, CD8+T and NKs. Furthermore, the immunocompetence of the TB.HSP70-CTL fusion protein was stronger than that of TB.HSP70/CTL complex. The HBV specific killing rate was 28.9%, the CD8+T cell population was 43.9% and the NKs was 13.6% in splenocytes of immunized mice with TB.HSP70-CTL fusion protein. The CTL peptide alone was capable of generating weak CTL lysis. The TB.HSP70 showed almost no target cell killing. Conclusion The results demonstrate that TB.HSP70 may be used as a new adjuvant carrier to improve the immunogenicity of short CTL epitope and produce effective CTL response.