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日本血吸虫HGPRT重组抗原与不同佐剂联合应用对小鼠免疫保护作用的研究
【摘要】 目的： 观察日本血吸虫(大陆株)次黄嘌呤鸟嘌呤磷酸核糖转移酶(hypoxanthineguanine phosphoribosyltransferase, HGPRT)重组抗原(reSjc HGPRT)与ISA206或弗氏佐剂联合免疫对小鼠诱导抗日本血吸虫感染的保护作用。 方法： 雌性C57BL/6小鼠随机分为5组:reSjc HGPRT加ISA206佐剂免疫组、reSjc HGPRT加弗氏佐剂组、ISA206佐剂对照组、弗氏佐剂对照组和感染对照组。20 μg重组抗原和ISA206或弗氏佐剂乳化后小鼠项背部多点皮下注射,共免疫3次,每次间隔2周。佐剂对照组小鼠仅注射ISA206或弗氏佐剂和生理盐水,感染对照组不注射任何重组抗原或生理盐水。于末次免疫后3周,每只小鼠经腹部皮肤感染(30±1)条日本血吸虫尾蚴,6周后剖杀小鼠,门脉灌注收集成虫,计数成虫数、雌雄合抱数和小鼠肝组织虫卵数。在免疫前、攻击感染前和小鼠剖杀前分别采血并分离血清,用ELISA检测血清中特异性IgG抗体。 结果： 重组抗原加ISA206佐剂或弗氏佐剂免疫组均诱导小鼠产生特异性IgG抗体应答,与感染对照组和弗氏佐剂对照组相比,差异有统计学意义(P<0.05);诱导小鼠产生的减虫率、减雌雄合抱率和肝组织减卵率分别为53.7%、59.3%、44.9%和43.3%、44.1%、33.0%,与感染对照组和2种佐剂对照组相比均有统计学意义(P<0.05)。结论: reSjc HGPRT与ISA206或弗氏佐剂联合免疫,可诱导小鼠产生抗血吸虫感染保护作用。
【关键词】 日本血吸虫; 次黄嘌呤鸟嘌呤磷酸核糖转移酶重组抗原; 佐剂; 免疫保护性
Protective immunity induced by recombinant Schistosoma hypoxanthineguanine
phosphoribosyltransferase with different adjuvants in mice医学 全在.线提供www.lindalemus.com
HU Yuan, SHEN Yujuan, CAO Jianping, XU Yuxin, LU Weiyuan,
ZHOU Hejun, ZHANG Jing, LI Xiaohong,QUAN Hong, LIU Shuxian
(National Institute of Parasite Disease, Chinese Center for Disease Control and Pevention, Key Laboratory of Parasite and Vector Biology,MOH, WHO Collaborating Center of Malaria, Schistosomiasis and Filariasis,Shanghai 200025,China)
[Abstract] Objective: To investigate the protective immunity of recombinant hypoxanthineguanine phosphoribosyltransferase of Schistosoma japonicum(reSjc HGPRT)plus Montanide ISA 206 or Freund’s adjuvants in mice. Methods： Female C57BL/6 mice were randomly divided into five groups, reSjc HGPRT plus Montanide ISA 206 adjuvant, reSjc HGPRT plus Freund’s adjuvant, two adjuvant control groups and challenged control group. In Montanide ISA 206 adjuvant or two groups of reSjc HGPRT plus Freund’s adjuvant, each mouse was immunized subcutaneously with 20 μg reSjc HGPRT emulsified in Montanide ISA 206 or FCA/FIA, respectively, while in two adjuvant control group, each mouse was injected subcutaneously with sterile normal saline emulsified in FCA/FIA or Montanide ISA 206 respectively. For challenged control group, mice were not treated with recombinant antigen or adjuvant. All mice were vaccinated for three times in an interval of 2 weeks. Two weeks after final immunization, each mouse was challenged with(30±1) cercariae of S. japonicum. At the sixth week after challenged, all mice were sacrificed and the number of worms, pairs of worm and eggs in the livers were counted. The sera collected respectively from mice before immunized, challenged and killed were identified by ELISA assay to detect specific antiSjcHGPRT IgG antibody. Results: The levels of specific IgG antibody in two immunized groups with reSjc HGPRT was higher than that of challenged control group. Also,compared with the challenged control, the worm reduction rates, worm in pairs rates and egg reduction rates in mice immunized with reSjc HGPRT plus Montanide ISA 206 adjuvant and plus Freund’s adjuvant were 53.7%, 59.3%, 44.9% and 43.3%, 44.1%, 33.0%, respectively(P<0.05). Conclusion: A better immune protection could be obtained in mice immunized with recombinant antigen plus plus Montanide ISA 206 or Freund’s adjuvants against S. japonicum.
[Key words] Schistosoma japonicum; reSjc HGPRT; adjuvant; immune protection
日本血吸虫病是严重危害人类健康的重要寄生虫病之一,目前尚无有效的预防措施。而抗血吸虫疫苗能够增强人群的抗血吸虫感染和再感染的能力,是长期综合防治血吸虫病的重要补充措施。日本血吸虫的生活史复杂,特别是由于其与宿主长期共进化,导致日本血吸虫能够逃避宿主的免疫攻击。故寻找合适的疫苗候选分子和选择适当的佐剂是疫苗研究的重点之一。
次黄嘌呤鸟嘌呤磷酸核糖转移酶(hypoxanthineguanine phosphoribosyltransferase, HGPRT)是一种与调控细胞核苷酸代谢有关的酶,在血吸虫体内有很高的活性,为血吸虫生长发育所必需。本研究在对日本血吸虫大陆株HGPRT编码基因克隆和表达取得成功的基础上[1],分别采用弗氏佐剂、Montanide ISA 206佐剂与重组蛋白乳化后免疫C57BL/6小鼠,并攻击感染血吸虫,探讨该重组蛋白(reSjc HGPRT)与佐剂联合抗血吸虫感染的保护作用。
1 材料与方法
1.1 材 料
实验用日本血吸虫大陆株感染的阳性钉螺由本所钉螺室提供,以常规方法逸得尾蚴。雌性6周龄的C57BL/6小鼠52只,由中国科学院上海实验动物中心提供。重组质粒pET28aSjc HGPRT的BL21 由本室构建。
1.2 重组抗原reSjc HGPRT的制备和纯化
