机构地区:[1]北京生物制品研究所第四研究室,100024 [2]北京协和医学院基础学院病原生物学系
出 处:《中华微生物学和免疫学杂志》2011年第12期1117-1123,共7页Chinese Journal of Microbiology and Immunology
基 金:国家863计划重大专项(2006AA02A224);传染病防治重大专项(2008ZX10004-015)
摘 要:目的 研究弗氏佐剂及3种人用佐剂[Al(OH)3、Montanide ISA720、Montanide ISA51]对重组多表位蛋白M.RCAg-1(malaria random constructed antigen-1)在BALB/c小鼠及新西兰大白兔体内产生免疫效果的影响.方法 以弗氏佐剂、Al( OH)3佐剂、Montanide ISA720以及MontanideISA51佐剂分别与M.RCAg-1蛋白进行配伍,分别免疫BALB/c小鼠及新西兰白兔;采用ELISA法检测M.RCAg-1蛋白及其所组成各个表位特异的血清IgG含量;间接荧光免疫试验分析不同配伍组免疫血清对天然疟原虫抗原的识别;酶联免疫斑点试验(ELISPOT)检测特异性鼠T淋巴细胞的活化;另借助生物传感器评价抗原与不同佐剂配伍组血清的亲和力,并用体外生长抑制试验(GIA)对恶性疟原虫的体外生长的影响进行评价.结果 不同佐剂可诱发不同水平的抗体滴度,Montanide ISA51佐剂配伍组的效果与弗氏佐剂组最为接近,并可使蛋白包含的11个表位肽能都较好诱发出特异性抗体,可有效激发以IFN-γ为主的细胞免疫应答,而且Montanide ISA51佐剂组免疫产生的抗体和M.RCAg-1蛋白质的亲和力高于其他两种佐剂;而Montanide ISA720佐剂和Al(OH)3佐剂免疫组不能诱发小鼠产生显著的IFN-γ反应(P>0.05).Montanide ISA51配伍蛋白质免疫产生的IgG在浓度为2mg/ml时对3D7和Dd2的抑制率分别为60%和100%,Al(OH)3佐剂组免疫兔血清IgG体外抑虫率较低(10%左右),而Montanide ISA720佐剂免疫组血清不能有效抑制疟原虫生长.结论 通过比较3种人用佐剂和弗氏佐剂对BALB/c小鼠和新西兰兔的免疫辅助效能显示,Montanide ISA51佐剂能很好的辅助M.RCAg-1蛋白疫苗诱发动物体内的体液免疫和细胞免疫应答,可做为疫苗的候选佐剂之一.Objective To detect the difference of cytokines and antibodies productions by immunologic system from mice and rabbits vaccinated with the M.RCAg-1 chimeric protein,expressed in E.coli,formulation with different adjuvants,including Freund's adjuvant and three clinically acceptable adjuvants,namely,Al(OH)3,Montanide ISA720 and Montanide ISA51.Methods Six weeks female BALB/c mice were vaccinated with recombinant protein formulated with different adjuvants through intranasal.Serum were collected to detect specific antibodies of M.RCAg-1 and individual Epitope by ELISA ; natural parasite antigen was recognized by indirect immunofluorescence assay; mouse specific T lymphocyte activation was detected by enzyme-linked immunosorbent spot test (ELISPOT) ; Affinity assay between protein and immune IgG of rabbits with the biosensor,and the growth of Plasmodiumfalciparum in vitro to evaluate by growth inhibition assay(GIA).Results Different formulation can induce different levels of antibody titers,the effection of ISA51 adjuvant was most closely with Freund's adjuvant,and can induce a higher specific antibody of 11 epitopes within proteins,can effectively stimulate cellular immune response based on the IFN-γ,to avidity Montanide ISA51 adjuvant immune antibodies and M.RCAg-1 protein affinity than the other two adjuvants;and Montanide ISA720 adjuvants and Al (OH)3 adjuvant group in mice can't induce a significant IFN-γresponse(P〉0.05).On avidity assay,the Montanide ISA51 formulation group was better than the other two adjuvants; and Montanide ISA720 and Al (OH)3 adjuvant formulation group can't induce a significant IFN-γresponse in mice(P〉0.05) ; the inhibition rates were 60% and 100% in 3D7 and Dd2 Plasmodium falciparum at a concentration of 2 mg/ml IgG by Montanide ISA51 formulated protein,and IgG of Al( OH)3 formulation could not effectively inhibit the in vitro growth of Plasmodium falciparum( 10% ),while IgG of Montanide ISA720 formulation could not inhibit growth of par
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