汉滩病毒核蛋白纯化及其免疫学特性的研究

Purification and immunological characteristics of HTNV NP expressed by the recombinant baculovirus

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 10 0LD50 76 118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 5 0× 10 3,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∶2 5 6 0 (IFAT)和 1∶2 0 480 (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?

Objective To investigate nucleocapsid protein (NP) of Hantaan virus (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∶2?560 by IFAT and 1∶20?480 by ELISA respectively), but no neutralizing antibody was found. Freud′s adjuvant 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.