CAJ | 학술논문

目的：在体外研究结核杆菌热休克蛋白70（TB ．HSP70）作为乙型肝炎病毒（HBV）核心抗原（HBcAg）细胞毒性T 淋巴细胞表位肽载体，诱导 HBV 特异性免疫应答。方法应用毕赤酵母分泌表达 HSP70（P1）、HSP70-HBcAg （18-27）（P2）、HSP70-PreS2B （18-24）-PreS2Th（37-53）-HBcAg（18-27）（P3），用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和蛋白免疫印迹法鉴定各重组蛋白的表达。体外考察重组蛋白（P1、P2、P3）对慢性乙型肝炎外周血来源的树突状细胞和淋巴细胞的作用，流式细胞术评估树突状细胞的成熟，酶联免疫吸附测定法检测细胞因子白细胞介素（IL ）-12p70、IL-1β、肿瘤坏死因子-α（T N F-α）、干扰素-γ（IFN-γ）分泌情况，TdR-3H掺入法检测淋巴细胞的增殖情况，应用经典51 Cr法检测重组蛋白诱导 HBV特异性细胞毒活性。结果重组蛋白P1、P2、P3构建成功，P1、P2、P3能诱导树突状细胞成熟，上调 CD1a、CD40、CD86表达，释放 T h1型细胞因子 IL-12p70、IL-1β和 TNF-α，P3最能有效激活自体淋巴细胞成为细胞毒活性细胞，促进淋巴细胞增殖和IFN-γ的释放，而单独的HSP70无杀伤活性。结论 TB ．HSP70可作为HBV HBcAg细胞毒性T 淋巴细胞表位肽载体，提高T 淋巴细胞表位肽的免疫原性，且含有B、T h表位的P3能更好地激活HBV特异性免疫应答。
목적：재체외연구결핵간균열휴극단백70（TB ．HSP70）작위을형간염병독（HBV）핵심항원（HBcAg）세포독성T 림파세포표위태재체，유도 HBV 특이성면역응답。방법응용필적효모분비표체 HSP70（P1）、HSP70-HBcAg （18-27）（P2）、HSP70-PreS2B （18-24）-PreS2Th（37-53）-HBcAg（18-27）（P3），용십이완기류산납-취병희선알응효전영화단백면역인적법감정각중조단백적표체。체외고찰중조단백（P1、P2、P3）대만성을형간염외주혈래원적수돌상세포화림파세포적작용，류식세포술평고수돌상세포적성숙，매련면역흡부측정법검측세포인자백세포개소（IL ）-12p70、IL-1β、종류배사인자-α（T N F-α）、간우소-γ（IFN-γ）분비정황，TdR-3H참입법검측림파세포적증식정황，응용경전51 Cr법검측중조단백유도 HBV특이성세포독활성。결과중조단백P1、P2、P3구건성공，P1、P2、P3능유도수돌상세포성숙，상조 CD1a、CD40、CD86표체，석방 T h1형세포인자 IL-12p70、IL-1β화 TNF-α，P3최능유효격활자체림파세포성위세포독활성세포，촉진림파세포증식화IFN-γ적석방，이단독적HSP70무살상활성。결론 TB ．HSP70가작위HBV HBcAg세포독성T 림파세포표위태재체，제고T 림파세포표위태적면역원성，차함유B、T h표위적P3능경호지격활HBV특이성면역응답。
Objective To investigate the effect of mycobacterium tuberculosis heat shock protein 70(TB .HSP70) as an adjuvant carrier on stimulating hepatitis B virus (HBV) core antigen(HBcAg)specific immune response to an accompanying cytotoxic T lym-phocytes epitope peptide from HBV core antigen in vitro .Methods Recombinant proteins HSP70(P1)、HSP70-HBcAg(18-27) (P2)、HSP70-PreS2B (18-24)-PreS2Th(37-53)-HBcAg(18-27)(P3) were expressed in methylotropic yeast Pichia pastoris GS115 . The expression of recombinant proteins was identified by SDS-PAGE and Western blot .The effect of recombinant proteins on den-dritic cell and lymphocytes of chronic HBV infection volunteers was investigated in vitro .The maturation of dendritic cell was meas-ured by flow cytometry ;the secretion of Th1 cytokines such as IL-12p70 ,IL-1β,TNF-αand IFN-γ was measured by ELISA ;the proliferation of lymphocytes was measured by TdR-3H ;the HBV-spesific cytotoxic activity was measured by the classic 51 Cr .Re-sults The recombinant proteins (P1 ,P2 ,P3) were constructed successfully .P1 ,P2 ,P3 could activate dendritic cell from chronic HBV infection volunteers by upregulation CD1a ,CD40 ,CD86 and production Th1 cytokines such as IL-12p70 ,IL-1β and TNF-α. Especially P3 could better induce autologous T cells to generate HBV specific cytotoxic T lymphocytes response ,activate the prolif-eration of lymphocytes and release IFN-γeffectively .However ,the recombinant HSP70 showed no target cell killing and could not induce immune response effectively .Conclusion TB .HSP70 can be used as an adjuvant carrier to stimulate HBV specific immune response to an accompanying cytotoxic T lymphocytes epitope peptide from HBV core antigen ,and enhance immunogenicity of the cytotoxic T lymphocytes epitope peptide .The P3 with B-and T-epitope can activate the HBV specific immune response effectively .