Objective To investigate nucleocapsid protein (NP) of Hantaanvirus (HTNV) and its immunological characteristics for a new subunit vaccine probably prepared. Methods The recombinant baculovirus, AcVHans containing S segment of HTNV, was generated by cotransfection of Sf9 cells with recombinant plasmid (PACHanS) and Bsu361-linearized AcVEPA DNA. The expression NP and wide-type NP were purified by immunoaffinity chromatography and confirmed by SDS-PAGE and Western blotting. The purified proteins were used to immunize BALB/c mice and M.unguiculatus; the specific antibodies in sera were detected by IFAT, ELISA, HA (hemagglutination activity), HI (hemagglutination-inhibition test), and NT (neutralization test). The lymphocyte transformation (3H-TdR incorporation) was used to test specific cellular immune response to HTNV. M.unguiculatus immunized by purified NP were used to do animal protection experiment. The role of peritoneal macrophages from the immunized mice in HTNV infection was studied with MTT colorimetric assay and IFAT. Results The purified protein showed one single band with 50,000 dalton on SDS-PAGE, and were confirmed to be HTNV NP by Western blotting. High-titer antibodies were detected in the sera of immunized BALB/c mice (1∶2560 by IFAT and 1∶20480 by ELISA respectively), but no neutralizing antibody was found. Freud′sadjuvant was better than Al(OH)3 adjuvant. The HA and the HI could be detected in immune sera. The specific cellular immune response to HTNV was observed in the immunized mice by lymphocyte transformation test. The SSI (specific stimulus indication) was more than 2.0. There was no obvious difference between Apodemus-type virus and Rattus-type virus. The immunized M.unguiculatus could resist HTNV challenge. MTT colorimetric assay showed that HTNV could inhibit the activities of peritoneal macrophages of mice that were not immunized by purified NP. The resistant rate was 47.6% to 76-118 and 46.6% to L99. The immune groups did not show obvious difference. We also found that macrophages of immunized mice that were infected by HTNV did not show any appearance of viral antigen in IFAT, while macrophages of control group were resulted in a reduction of cell viability and appearance of viral antigens. Conclusions The recombinant NP and the wild-type NP have same biological activities and show good immune effects. On the NP of HTNV locate HA epitopes, HI epitopes, and the antigenic epitopes which can induce the specific cellular immune responses, but no neutralizing epitopes. The NP of HTNV have immune protection effects, and the macrophages of NP immunized mice can resist HTNV infection specifically.%目的 研究汉滩病毒核蛋白及其免疫学特性，研制新一代的肾综合征出血热(HFRS)亚单位疫苗。方法 用亲和层析法纯化重组杆状病毒表达的汉滩病毒核蛋白及野生汉滩病毒76-118的核蛋白，以聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹实验(Westernblot)鉴定纯化蛋白。用纯化蛋白免疫BALB/c小鼠，分别以福氏佐剂(FCA)和氢氧化铝[Al(OH)3]为佐剂。以免疫荧光法(IFAT)及酶联免疫法(ELISA)检测抗体滴度；用微量细胞病变中和试验检测中和抗体，用血凝试验检测血凝活性，用血凝抑制试验检测血凝抑制活性；用淋巴细胞转化试验(3H-TdR掺入法)测定免疫小鼠脾淋巴细胞对汉坦病毒76-118株(野鼠型)和L99(家鼠型)的细胞免疫应答；动物保护试验以长爪沙鼠为感染动物模型，用纯化的核蛋白进行免疫，再用100LD5076-118进行攻击，然后以免疫荧光法观察鼠肺切片；用MTT比色法观察免疫小鼠巨噬细胞接种汉滩病毒后细胞活力的变化并用免疫荧光法检测免疫小鼠巨噬细胞中的病毒抗原。结果 SDS-PAGE显示单一条带，相对分子质量均为50×103,Westernblot证明为汉滩病毒核蛋白。小鼠免疫血清抗体滴度分别为1∶2560(IFAT)和1∶20480(ELISA)。福氏佐剂比氢氧化铝佐剂能更好地加强抗原的免疫原性。小鼠免疫血清用微量细胞病变中和试验未检测到中和抗体。在血凝试验中发现免疫小鼠血清有血凝活性，在血凝抑制试验中发现免疫小鼠血清有微弱的血凝抑制活性。用3H-TdR掺入法结果表明，免疫小鼠的脾淋巴细胞对两型病毒均有特异性增殖反应(特异性刺激指数SSI>2.0)，且对两型病毒的增殖性反应无明显差别。在动物保护试验中，鼠肺切片结果表明核蛋白能使长爪沙鼠获得对病毒攻击的保护。MTT法表明汉坦病毒对未免疫对照组巨噬细胞的抑制率分别达47.6%(76-118)和46.6%(L99)，而对蛋白免疫组巨噬细胞的抑制作用不明显。巨噬细胞的免疫荧光检测结果表明，用纯化核蛋白免疫的小鼠巨噬细胞，其病毒抗原检测全部阴性，而在对照组中可观察到汉滩病毒典型的特异性荧光颗粒。结论 重组核蛋白和野生核蛋白具有相同的生物学活性，两者均具有良好的免疫原性。其上有血凝活性位点、血凝抑制活性位点及引起细胞免疫的抗原位点，但无中和活性位点。动物保护试验证明其具有免疫保护作用，经其免疫的小鼠巨噬细胞具有特异性的抗病毒感染作用。
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