CAJ | 학술논문

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.
목적 연구불씨좌제급3충인용좌제[Al(OH)3、Montanide ISA720、Montanide ISA51]대중조다표위단백M.RCAg-1(malaria random constructed antigen-1)재BALB/c소서급신서란대백토체내산생면역효과적영향.방법 이불씨좌제、Al( OH)3좌제、Montanide ISA720이급MontanideISA51좌제분별여M.RCAg-1단백진행배오,분별면역BALB/c소서급신서란백토；채용ELISA법검측M.RCAg-1단백급기소조성각개표위특이적혈청IgG함량；간접형광면역시험분석불동배오조면역혈청대천연학원충항원적식별；매련면역반점시험(ELISPOT)검측특이성서T림파세포적활화；령차조생물전감기평개항원여불동좌제배오조혈청적친화력,병용체외생장억제시험(GIA)대악성학원충적체외생장적영향진행평개.결과 불동좌제가유발불동수평적항체적도,Montanide ISA51좌제배오조적효과여불씨좌제조최위접근,병가사단백포함적11개표위태능도교호유발출특이성항체,가유효격발이IFN-γ위주적세포면역응답,이차Montanide ISA51좌제조면역산생적항체화M.RCAg-1단백질적친화력고우기타량충좌제；이Montanide ISA720좌제화Al(OH)3좌제면역조불능유발소서산생현저적IFN-γ반응(P＞0.05).Montanide ISA51배오단백질면역산생적IgG재농도위2mg/ml시대3D7화Dd2적억제솔분별위60%화100%,Al(OH)3좌제조면역토혈청IgG체외억충솔교저(10%좌우),이Montanide ISA720좌제면역조혈청불능유효억제학원충생장.결론 통과비교3충인용좌제화불씨좌제대BALB/c소서화신서란토적면역보조효능현시,Montanide ISA51좌제능흔호적보조M.RCAg-1단백역묘유발동물체내적체액면역화세포면역응답,가주위역묘적후선좌제지일.
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 parasite in vitro.Conclusion By comparing three clinically acceptable adjuvants and Freund's adjuvant in BALB/c mice and New Zealand rabbit,Montanide ISA51 adjuvants can be acceptable formulated M.RCAg-1 protein induced humoral and cellular immune responses,can be used as one of the candidate adjuvants.