本文選題：惡性瘧原蟲 + 表位改組��； 參考：《中國協(xié)和醫(yī)科大學(xué)》2010年博士論文

【摘要】： 摘要 瘧疾是世界上最嚴重的傳染病之一,2008年有2.43億人感染瘧原蟲,約86.3萬人因患瘧疾死亡,而惡性瘧原蟲是造成嚴重危害的主要“元兇”。近年來,雖然在“瘧疾控制”方面取得了顯著的成績,但對于預(yù)防性和治療性瘧疾疫苗的需求仍十分迫切。目前,針對瘧原蟲生活史復(fù)雜,抗原的表達具有階段特異性、高度變異性等特點,亞單位、多表位嵌合疫苗成為抗惡性瘧疫苗的研究熱點。 前期研究中,本實驗室發(fā)明了“表位改組”技術(shù),利用其構(gòu)建了具有極高表位連接多樣性的多表位基因文庫,并篩選出了高免疫原性和體外抑制瘧原蟲生長能力的第一代多表位嵌合抗原M.RCAg-1,證實了“表位改組”技術(shù)的可行性。但是,由于其N端帶有載體來源的43肽以及6×His蛋白標簽,雖然能夠用腸激酶(EK)切除,但成本過高,限制了M.RCAg-1的研究價值。 本研究在前期工作的基礎(chǔ)上,進行了一系列改進,包括：選擇了12個以惡性瘧原蟲紅內(nèi)期主要的疫苗候選抗原為主的15個B細胞和Th細胞表位；在表位連接處引入特定的間隔序列；在多表位抗原的N、C端固定引入抗原側(cè)翼序列FN和FC；所有多表位抗原序列均為瘧原蟲來源,不含任何載體肽和蛋白標簽,符合國家《人用重組DNA制品質(zhì)量控制技術(shù)指導(dǎo)原則》的標準規(guī)范。構(gòu)建并篩選得到了第二代多表位嵌合抗原疫苗。與此同時,通過對多表位嵌合抗原序列(表位)組成、二級結(jié)構(gòu)、免疫原性和瘧原蟲體外生長抑制率(GIA)水平的對比分析,初步探討了幾個參數(shù)間的關(guān)系。此外,建立了以C端固定肽的特異性多克隆IgY抗體為配基的免疫親和層析,為文庫中多表位嵌合抗原的高效純化探索了新的途徑。 主要取得以下進展： 1.成功構(gòu)建并得到具有高免疫原性和表位連接多樣性的第二代多表位嵌合基因文庫,同時其小鼠免疫血清對培養(yǎng)惡性瘧原蟲天然蛋白具有識別多樣性。 2.從基因文庫中篩選得到高免疫原性嵌合抗原D10,免疫大白兔后,總IgG和特異性IgG的GIA水平分別為47%和67%,高于陽性對照抗原MSP1抗體的44%。此外,對72份疫區(qū)患者血清的篩查發(fā)現(xiàn),對D10抗原的識別率達到67%,且與患者蟲血率顯著負相關(guān),提示D10可能與惡性瘧疾保護有關(guān)。表明全序列為瘧原蟲來源的嵌合抗原D10,具有繼續(xù)優(yōu)化開發(fā)的價值。 3.我們發(fā)現(xiàn)多表位嵌合抗原二級結(jié)構(gòu)中的"Coil-Helix-Coil-Helix"結(jié)構(gòu)影響抗原的免疫原性；同時,也發(fā)現(xiàn)含有“連續(xù)4個Th細胞表位”結(jié)構(gòu)的多表位嵌合抗原具有更高的GIA水平。 4.證實了在多表位嵌入肽段抗體中,抑制性和促進性抗體的存在,并推斷其以級聯(lián)或協(xié)同的方式影響總抗體的GIA水平。 5.通過制備C端融合序列的特異性多克隆IgY抗體,建立了免疫親和純化文庫多表位嵌合抗原的新途徑。 6.證實了基于Hydroethidine染料的流式細胞術(shù),可以作為惡性瘧原蟲蟲血率及其生長周期的相對定量分析方法,并將其工作濃度優(yōu)化為10μg/ml。 本研究利用部分改進的“表位改組”技術(shù),構(gòu)建并篩選得到了具有較大開發(fā)價值的多表位嵌合抗原D10。同時,通過大量實驗數(shù)據(jù)證實了N、C端融合片段作為鑒定和免疫親和純化內(nèi)源標簽的實際應(yīng)用性,以及多表位嵌入肽段序列對多表位嵌合抗原免疫原性和其特異性抗體對瘧原蟲體外生長抑制率的影響。以上實驗結(jié)果為各類多表位嵌合抗原疫苗的設(shè)計提供了有意義的理論依據(jù)。
[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.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【引證文獻】

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1 吳巍;藺亞暉;馬瑞森;席玨敏;王恒;;電穿孔法提高惡性瘧疾DNA疫苗免疫原性[J];中國生物醫(yī)學(xué)工程學(xué)報;2011年01期

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本文編號：1911224