【作者】 曲久鑫；

【导师】 王恒； 张伟； 许雪梅；

【作者基本信息】 中国协和医科大学 ， 病原生物学， 2010， 博士

【摘要】 摘要疟疾是世界上最严重的传染病之一,2008年有2.43亿人感染疟原虫,约86.3万人因患疟疾死亡,而恶性疟原虫是造成严重危害的主要“元凶”。近年来,虽然在“疟疾控制”方面取得了显著的成绩,但对于预防性和治疗性疟疾疫苗的需求仍十分迫切。目前,针对疟原虫生活史复杂,抗原的表达具有阶段特异性、高度变异性等特点,亚单位、多表位嵌合疫苗成为抗恶性疟疫苗的研究热点。前期研究中,本实验室发明了“表位改组”技术,利用其构建了具有极高表位连接多样性的多表位基因文库,并筛选出了高免疫原性和体外抑制疟原虫生长能力的第一代多表位嵌合抗原M.RCAg-1,证实了“表位改组”技术的可行性。但是,由于其N端带有载体来源的43肽以及6×His蛋白标签,虽然能够用肠激酶(EK)切除,但成本过高,限制了M.RCAg-1的研究价值。本研究在前期工作的基础上,进行了一系列改进,包括：选择了12个以恶性疟原虫红内期主要的疫苗候选抗原为主的15个B细胞和Th细胞表位；在表位连接处引入特定的间隔序列；在多表位抗原的N、C端固定引入抗原侧翼序列FN和FC；所有多表位抗原序列均为疟原虫来源,不含任何载体肽和蛋白标签,符合国家《人用重组DNA制品质量控制技术指导原则》的标准规范。构建并筛选得到了第二代多表位嵌合抗原疫苗。与此同时,通过对多表位嵌合抗原序列(表位)组成、二级结构、免疫原性和疟原虫体外生长抑制率(GIA)水平的对比分析,初步探讨了几个参数间的关系。此外,建立了以C端固定肽的特异性多克隆IgY抗体为配基的免疫亲和层析,为文库中多表位嵌合抗原的高效纯化探索了新的途径。主要取得以下进展：1.成功构建并得到具有高免疫原性和表位连接多样性的第二代多表位嵌合基因文库,同时其小鼠免疫血清对培养恶性疟原虫天然蛋白具有识别多样性。2.从基因文库中筛选得到高免疫原性嵌合抗原D10,免疫大白兔后,总IgG和特异性IgG的GIA水平分别为47%和67%,高于阳性对照抗原MSP1抗体的44%。此外,对72份疫区患者血清的筛查发现,对D10抗原的识别率达到67%,且与患者虫血率显著负相关,提示D10可能与恶性疟疾保护有关。表明全序列为疟原虫来源的嵌合抗原D10,具有继续优化开发的价值。3.我们发现多表位嵌合抗原二级结构中的"Coil-Helix-Coil-Helix"结构影响抗原的免疫原性；同时,也发现含有“连续4个Th细胞表位”结构的多表位嵌合抗原具有更高的GIA水平。4.证实了在多表位嵌入肽段抗体中,抑制性和促进性抗体的存在,并推断其以级联或协同的方式影响总抗体的GIA水平。5.通过制备C端融合序列的特异性多克隆IgY抗体,建立了免疫亲和纯化文库多表位嵌合抗原的新途径。6.证实了基于Hydroethidine染料的流式细胞术,可以作为恶性疟原虫虫血率及其生长周期的相对定量分析方法,并将其工作浓度优化为10μg/ml。本研究利用部分改进的“表位改组”技术,构建并筛选得到了具有较大开发价值的多表位嵌合抗原D10。同时,通过大量实验数据证实了N、C端融合片段作为鉴定和免疫亲和纯化内源标签的实际应用性,以及多表位嵌入肽段序列对多表位嵌合抗原免疫原性和其特异性抗体对疟原虫体外生长抑制率的影响。以上实验结果为各类多表位嵌合抗原疫苗的设计提供了有意义的理论依据。更多还原

【Abstract】 Malaria is one of the most severe infectious diseases in the world,there are 243 million cases and 0.86million deaths caused by malaria in 2008,and Plasmodium falciparum is the most deadly species. Recent years,though marked progresses have been obtained in"Malaria Control",there is still an urgent demand of preventive and therapeutic vaccine.To overcome the problem of highly antigenic variation and stage specincity in P.falciparum,sub-unit or polyepitope chimeric vaccine is a promising strategy.In previous study,we generated a novel approach named"epitope shuffling".Using the approach,we constructed a polyepitope gene library with high diversity of epitope combination,and from which our first generation of polyeptitope vaccine designated M.RCAg-1 with high immunogenicity and in vitro antipalasite efficacy was obtained.However,the antigen’s further clinical study had been hampered by the redundancy of 43 amino acids from vector at N terminal and 6×His tag,though which could be cut off by Enterokinase with high cost.Based on the previous work,we have made several improvements,which includes:(1)15 B cell and Th cell epitopes from 12 main antigens of P.falciparum blood stage have been selected,(2)definite spacer sequences have been introduced between adjacent epitopes,(3)two parasite-derived fusion sequences FN and FC as flanking sequences have been fixed at N and C terminals of polyepitope antigen,(4)to meet the"national standard of DNA recombinant products",no vector peptides or tags are contained in the sequence.The second generation of polyepitope chimeric vaccine have been constructed and screened.Meanwhile,by comparative analysis,we have explored the relationship among epitope composition,second strueture,immunogenicity and in vitro antiparasite efficacy of the polyepitope antigens.Moreover,high efficient immunoaffinity purification of polyepitope antigens by IgY specific to FC has been investigated.The results are as follows:1. The second generation of polyepitope gene libraly with high immunogenicity and diversity of epitope combination had been successfully constructed.Besides,the immune serum from mice could recognize the native proteins of P.falciparum cultured in lab with high diversity. 2.The chimeric antigen named D10 with high immunogenicity was screened from the gene library. The results of GIA using total and specific IgG from rabbit were 47% and 67%,which were both higher than 44% of the result fiom positive control MSP1 antibody.On the other hand,D10 could be recognized by 67% of 72 samples of patients’ sera from epidemic area and the recognization was significantly negatively correlated with parasitemia of patients,which suggested that D10 could be correlated with the protection against P.falciparum.Based on these results,antigen D10, full sequences derived from P.falciparum,is worthy of further development.3."Coil-Helix-Coil-Helix"structure existed in the secondary structures of polyepitope antigens could reflect the immunogenicity.And the"4 successive Th cell epitope"structure contained polyepitope antigens had higher in vitro antiparasite efficacy of antibodies than others.4. Inhibiting and enhancing antibodies had been proved to be existed in the total antibodies against polyepitope fragments of chimeric antigens,which would probably affect the in vitro antiparasite efficacy of total antibodies by synergism mechanism.5. High efficiency of chimeric antigens’ purification by immunoaffinity chromatography with polyclonal IgY antibodies specific to C-terminal fusion polypeptides had been confirmed.6. Hydroethidine-based flow cytometry,as a relative quantitative method,could be applied in the analysis of parasitemia and life cycle in the culture of P.falciparum,and the working concentration of Hydroethidine had been optimized with 10μg/ml.In conclusion,the second generation of polyepitope chimeric antigen D10 had been constructed and screened by the improved"epitope shuffling",which would be worthy of further development.And the present results indicated that N and C terminal fusions could be used as"endogenous tag"for the identification and immunoaffinity purification of the antigens.Besides,the assumption had been confirmed that polyepitope fragments could impact the immunogenicity and in vitro antiparasite efficacy of the chimeric antigens.These results would be valuable for the design and optimization of various kinds of polyepitope chimeric vaccines.更多还原