日本血吸虫重组IAP蛋白的免疫保护效果评估

摘要

To evaluate the protective immunity induced by the inhibitor of apoptosis protein of Schistosoma japonicum (rSjIAP) in BALB/c mice, SjIAP gene was amplified in PCR and subcloned into the plasmid pET-28a(+) to construct a recombinant expression plasmid pET-28a(+)-SjIAP. The recombinant protein SjIAP was expressed in E.coli. The purified recombinant protein was used to immunize New Zealand rabbit to produce polyclonal antibodies. BALB/c mice were immunized with the purified recombinant SjIAP emulsified in Freund's adjuvant. The levels of specific anti-rSjIAP antibodies in serum samples of immunized animals and cytokines in culture supernatants of the spleen lymphocytes in response to the T cell mitogen rSjIAP were detected in ELISA. Then all mice were challenged with Schistosoma japonicum cercariae and terminated at thirty-eight days later. The rates of worm reduction and liver egg reduction were counted. Western blot analysis indicated that rSjIAP reacted with rabbit polyclonal antibodies. High levels of specific IgG and IgG subclass (IgG1, IgG2a, IgG2b and IgG4) antibodies as well as IFN-γ, IL-2 and IL-4 were detected in animal serum samples. The immunized mice showed worm reduction by 31.5%and liver egg reduction by 37.2%. The present study indicated that immunization of rSjIAP induced partial protection against schistosomiasis in BALB/c mice, suggesting that SjIAP might be a potential candidate for vaccine development.%为研究日本血吸虫凋亡蛋白抑制因子（inhibitor of apoptosis protein of Schistosoma japonicum, SjIAP）重组蛋白诱导BALB/c小鼠的免疫保护效果，利用PCR技术扩增SjIAP基因，构建重组表达质粒pET-28a（+）-SjIAP，诱导表达重组SjIAP蛋白，并利用重组蛋白制备兔源多克隆抗体血清。然后，选用SjIAP重组蛋白免疫BALB/c小鼠，利用ELISA检测免疫小鼠血清中的特异性抗体水平，以及免疫小鼠脾脏淋巴细胞在SjIAP重组蛋白刺激后产生的细胞因子水平。强化免疫后，将小鼠进行血吸虫尾蚴攻虫试验，感染38 d，进行剖杀，计算虫体减虫率及肝脏减卵率。Western blot结果表明本研究制备的兔源多抗血清能特异性识别SjIAP重组蛋白。ELISA检测表明免疫SjIAP重组蛋白可诱导较高水平的IgG及IgG亚型（IgG1、IgG2a、IgG2b、IgG3）抗体和IFN-γ、IL-2及IL-4细胞因子。动物试验表明，免疫SjIAP重组蛋白的小鼠与PBS组相比分别获得了31.5%的减虫率和37.2%的肝脏减卵率。免疫SjIAP重组蛋白能诱导小鼠获得一定减虫和减卵保护效果，提示血吸虫凋亡蛋白抑制因子可作为抗血吸虫病的疫苗候选分子。