第二代恶性疟原虫多表位人工抗原疫苗的构建和筛选

发布时间：2018-05-19 18:45

本文选题：恶性疟原虫 + 表位改组　；参考：《中国协和医科大学》2010年博士论文

【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。
[Abstract]:abstract
Malaria is one of the most serious infectious diseases in the world. In 2008, 243 million people were infected with malaria parasites, about 863 thousand people died of malaria, and Plasmodium falciparum was the major "major culprit". In recent years, although significant achievements have been made in "malaria control", the demand for preventive and therapeutic malaria vaccines still remains. At present, in view of the complexity of the life history of Plasmodium, the expression of antigen has the characteristics of stage specificity and high variability, subunit, multi epitope chimeric vaccine has become a hot spot in the research of anti falciparum malaria vaccine.
In the previous study, our laboratory invented the "epitope reorganization" technique, using it to construct a multi epitope gene library with high epitope diversity, and screened the first generation of multi epitope chimeric antigen M.RCAg-1 with high immunogenicity and in vitro inhibition of Plasmodium growth. Because its N terminal contains 43 peptide derived from the carrier and 6 x His protein tag, although it can be removed by EK, the cost is too high, which limits the research value of M.RCAg-1.
On the basis of previous work, a series of improvements were carried out, including: selecting 12 B cells and Th cell epitopes based on the main vaccine candidate antigens of Plasmodium falciparum during the period of Plasmodium falciparum; introducing specific spacer sequences at the epitope junctions; the introduction of the antigen flanking sequence FN and FC at the N of the multi epitope antigen and the C end of the epitope. The multi epitope antigen sequence was the source of Plasmodium, without any carrier peptide and protein label, conformed to the National Standard Specification for the guiding principle of quality control technology for human recombinant DNA products. The second generation of multi epitope chimeric antigen vaccine was constructed and screened. At the same time, the two level junctions were composed of the multiple epitope antigen sequence (epitopes). Contrastive analysis of the immunogenicity and the growth inhibition rate (GIA) of Plasmodium in vitro, the relationship between several parameters was preliminarily discussed. In addition, an immunoaffinity chromatography with specific polyclonal IgY antibody of the C terminal fixed peptide was established, and a new approach was explored for the efficient purification of polyepitope antigen in the library.
The main progress has been made as follows:
1. the second generation multi epitope chimeric gene library with high immunogenicity and epitope diversity was constructed successfully, and the immune sera of the mice had the identification diversity for the cultivation of the natural protein of Plasmodium falciparum.
2. the high immunogenic chimeric antigen D10 was screened from the gene library. After immunization, the GIA level of the total IgG and specific IgG was 47% and 67% respectively, which was higher than the 44%. of the positive control antigen MSP1 antibody. The screening of the serum of the patients in the epidemic area was found to be 67% for the recognition rate of the D10 antigen and significantly negatively correlated with the blood rate of the patients. The expression of D10 may be related to the protection of falciparum malaria, indicating that the chimeric antigen D10, which is the source of malaria parasite, has the value of continuous optimization.
3. we found that the "Coil-Helix-Coil-Helix" structure in the multi epitope chimeric antigen two structure affects the immunogenicity of the antigen; at the same time, it is also found that the multi epitope chimeric antigen containing "4 consecutive Th cell epitopes" has a higher level of GIA.
4. confirmed the presence of inhibitory and promotive antibodies in the multi epitope embedded peptide antibody and infers that it affects the GIA level of the total antibody in a cascade or synergistic manner.
5. through the preparation of specific polyclonal IgY antibodies from C terminal fusion sequences, a new immunoaffinity purified library multi epitope chimeric antigen was established.
6. the flow cytometry based on Hydroethidine dyes was proved to be a relative quantitative analysis method for the blood rate and growth cycle of Plasmodium falciparum and to optimize its working concentration to 10 mu g/ml..
In this study, we used some improved "epitopes" techniques to construct and screen the multiple epitope chimeric antigen D10. with greater development value. Through a large number of experimental data, we confirmed the practical application of N, C end fusion fragments as identification and immuno affinity purified endogenous tags, as well as multiple epitopes sequence sequences for multiple epitopes. The effect of chimeric antigen immunogenicity and its specific antibody on the growth inhibition rate of Plasmodium in vitro. The above experimental results provide a meaningful theoretical basis for the design of various epitope chimeric antigens.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【引证文献】