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日本血吸蟲HGPRT重組抗原與不同佐劑聯(lián)合應(yīng)用對小鼠免疫保護作用的研究
【摘要】 目的： 觀察日本血吸蟲(大陸株)次黃嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶(hypoxanthineguanine phosphoribosyltransferase, HGPRT)重組抗原(reSjc HGPRT)與ISA206或弗氏佐劑聯(lián)合免疫對小鼠誘導(dǎo)抗日本血吸蟲感染的保護作用。 方法： 雌性C57BL/6小鼠隨機分為5組:reSjc HGPRT加ISA206佐劑免疫組、reSjc HGPRT加弗氏佐劑組、ISA206佐劑對照組、弗氏佐劑對照組和感染對照組。20 μg重組抗原和ISA206或弗氏佐劑乳化后小鼠項背部多點皮下注射,共免疫3次,每次間隔2周。佐劑對照組小鼠僅注射ISA206或弗氏佐劑和生理鹽水,感染對照組不注射任何重組抗原或生理鹽水。于末次免疫后3周,每只小鼠經(jīng)腹部皮膚感染(30±1)條日本血吸蟲尾蚴,6周后剖殺小鼠,門脈灌注收集成蟲,計數(shù)成蟲數(shù)、雌雄合抱數(shù)和小鼠肝組織蟲卵數(shù)。在免疫前、攻擊感染前和小鼠剖殺前分別采血并分離血清,用ELISA檢測血清中特異性IgG抗體。 結(jié)果： 重組抗原加ISA206佐劑或弗氏佐劑免疫組均誘導(dǎo)小鼠產(chǎn)生特異性IgG抗體應(yīng)答,與感染對照組和弗氏佐劑對照組相比,差異有統(tǒng)計學(xué)意義(P<0.05);誘導(dǎo)小鼠產(chǎn)生的減蟲率、減雌雄合抱率和肝組織減卵率分別為53.7%、59.3%、44.9%和43.3%、44.1%、33.0%,與感染對照組和2種佐劑對照組相比均有統(tǒng)計學(xué)意義(P<0.05)。結(jié)論: reSjc HGPRT與ISA206或弗氏佐劑聯(lián)合免疫,可誘導(dǎo)小鼠產(chǎn)生抗血吸蟲感染保護作用。
【關(guān)鍵詞】 日本血吸蟲; 次黃嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶重組抗原; 佐劑; 免疫保護性
Protective immunity induced by recombinant Schistosoma hypoxanthineguanine
phosphoribosyltransferase with different adjuvants in mice醫(yī)學(xué) 全在.線提供m.zxtf.net.cn
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
日本血吸蟲病是嚴重危害人類健康的重要寄生蟲病之一,目前尚無有效的預(yù)防措施。而抗血吸蟲疫苗能夠增強人群的抗血吸蟲感染和再感染的能力,是長期綜合防治血吸蟲病的重要補充措施。日本血吸蟲的生活史復(fù)雜,特別是由于其與宿主長期共進化,導(dǎo)致日本血吸蟲能夠逃避宿主的免疫攻擊。故尋找合適的疫苗候選分子和選擇適當(dāng)?shù)淖魟┦且呙缪芯康闹攸c之一。
次黃嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶(hypoxanthineguanine phosphoribosyltransferase, HGPRT)是一種與調(diào)控細胞核苷酸代謝有關(guān)的酶,在血吸蟲體內(nèi)有很高的活性,為血吸蟲生長發(fā)育所必需。本研究在對日本血吸蟲大陸株HGPRT編碼基因克隆和表達取得成功的基礎(chǔ)上[1],分別采用弗氏佐劑、Montanide ISA 206佐劑與重組蛋白乳化后免疫C57BL/6小鼠,并攻擊感染血吸蟲,探討該重組蛋白(reSjc HGPRT)與佐劑聯(lián)合抗血吸蟲感染的保護作用。
1 材料與方法
1.1 材 料
實驗用日本血吸蟲大陸株感染的陽性釘螺由本所釘螺室提供,以常規(guī)方法逸得尾蚴。雌性6周齡的C57BL/6小鼠52只,由中國科學(xué)院上海實驗動物中心提供。重組質(zhì)粒pET28aSjc HGPRT的BL21 由本室構(gòu)建。
1.2 重組抗原reSjc HGPRT的制備和純化
