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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 江伯倫(Bor-Luen Chiang) | |
dc.contributor.author | Szu-Wei Lin | en |
dc.contributor.author | 林思瑋 | zh_TW |
dc.date.accessioned | 2021-06-13T00:13:57Z | - |
dc.date.available | 2007-08-08 | |
dc.date.copyright | 2007-08-08 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-27 | |
dc.identifier.citation | Reference
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/28606 | - |
dc.description.abstract | 腸病毒為微小RNA病毒科的一員,每年造成台灣許多兒童的感染,臨床症狀有發燒、腦炎和手口足症等等,有時也會引起嚴重的神經性疾病而導致死亡,因此,為了降低降低感染率,疫苗的研發是很重要的策略。在抗原上,腸病毒71型的其中一個外鞘蛋白VP1被認為應該是引起免疫反應的關鍵抗原,因此本研究中選用VP1做為疫苗抗原。Flt3L是一種細胞激素,對很多免疫細胞的發育都有重要的作用,近年一些研究也指出,在週邊組織施打Flt3L可以增加樹突細胞(一種抗原呈現細胞)的數目,以及促進其成熟。所以,此一研究的目的是要利用Flt3L作為佐劑以增加抗原細胞呈現VP1的機會。我們實驗室之前已經做過實驗,同時給予小鼠Flt3L和VP1的基因可以引起VP1特異性免疫球蛋白A、G和M,在這次研究中,我們先將Flt3L和VP1的基因連在一起後再用質體致敏小鼠,並採用多次給予質體後給予一次蛋白的方式,然後以特異性免疫球蛋白的量以及其對於抑制病毒感染的能力做為指標; 另外,也直接混合Flt3L和VP1蛋白作為為疫苗,可同時比較及因疫苗與蛋白疫苗的優劣。結果發現合併使用Flt3L和VP1蛋白或利用Flt3L-VP1重組基因,都可以增加小鼠體內特異性抗體的產生,並能引發較強的黏膜免疫反應,降低感染的機會; 同時,我們認為Flt3L不只在免疫細胞發育時有關鍵的作用,同時也可以作為一個很好的佐劑,在疫苗發展上值得繼續研究。 | zh_TW |
dc.description.abstract | Enterovirus 71 infects many children and adults and and might result in mortality in Taiwan every year. It is important to develop vaccine for protection against enterovivus infection. Flt3 (fms-like tyrosine kinase 3) ligand is an important hematopoietic cytokine. Recent studies suggested that in vivo Flt3L treatment of mice and human volunteers resulted in dramatic increases in the number of functional dendritic cells in bone marrow, spleen, thymus, and various lymphoid tissues. As a result, we tried to apply Flt3L as the adjuvant for enhancing the immunogenecity of antigen. According to our previous result, we found that delivery of plasmids containing flt3l and plasmids containing vp1 to BALB/c mice could elicit serum VP1-specific antibodies. In this study, we tried to construct a fusion protein of adjuvant Flt3L and antigen VP1. In vitro functional assay showed that the fusion protein alone could stimulate dendritic cell differentiation and maturation as treatment of IL-4 and GM-CSF could. Therefore, we tried to enhance EV71 vaccine efficiency on BALB/c mice via intranasal delivery of fl-vp1 fusion gene and FL protein combined with VP1 protein.The results showed that both vaccine strategies could elicit high titer of specific antibodies. The neutralization titers of some experimental groups were better than positive groups and the antibodies in sera had protection against enterovirus infection. We also found that in DNA vaccines, the effect of fusion gene was better than individual genes and protein vaccines were more effective than DNA vaccines. Thus, it was suggested that Flt3L could be a good and potent vaccine adjuvant, probably more potential than cholera toxin in enterovirus vaccine development. In the future, we may apply Flt3L combined with virus-like particles or other enterovirus capsid proteins to search a better vaccine candidate. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T00:13:57Z (GMT). No. of bitstreams: 1 ntu-96-R94450002-1.pdf: 5573785 bytes, checksum: dff88b0459775b7e011ab9208a6ec8b3 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | Acknowledgement………………………………………………………………………i
中文摘要………………………………………………………………………………ii Abstract………………………………………………………………………………iii Contents………………………………………………………………………………iv Contents of figures and tables………………………………………………………vii Introduction……………………………………………………………………………1 1.1 Introduction to Enterovirus 71……………………………………………………1 1.1.1 Epidemiology of Enterovirus 71………………………………………………1 1.1.2 Clinical syndromes of Enterovirus 71 infection………………………………2 1.1.3 Structural and properties of Enterovirus 71……………………………………4 1.1.4 1.1.4 Antigenic determinant sites of Enterovirus 71…………………………………5 1.1.5 Infection, therapy and vaccine development. …………………………………6 1.2 Introduction to Flt3 ligand…………………………………………………………7 1.2.1 Receptor and ligand……………………………………………………………7 1.2.2 The biodistribution of Flt3 and Flt3 ligand……………………………………8 1.2.3 The synergy of flt3 ligand………………………………………………………8 1.2.4 The application of flt3 ligand in vaccine development…………………………9 1.3 Introduction to mucosal immunity………………………………………………11 1.3.1 Mucosa system…………………………………………………………………11 1.3.2 Mucosal effector immune response……………………………………………12 1.3.3 Immunization routes…………………………………………………………13 1.3.4. Adjuvants of mucosal vaccines………………………………………………14 1.4 Aim and purpose…………………………………………………………………15 Materials and methods………………………………………………………………17 2.1 Materials…………………………………………………………………………17 2.1.1 Vectors…………………………………………………………………………17 2.1.2 Animals………………………………………………………………………17 2.1.3 Cell lines………………………………………………………………………17 2.1.4 Broth, plate and medium of E.coli incubation…………………………………17 2.1.5 Medium, buffer and reagents of cell culture…………………………………18 2.1.6 Reagents of ELISA assay……………………………………………………19 2.1.7 Western blot……………………………………………………………………20 2.1.8 Buffers of protein purification…………………………………………………21 2.1.9 Others…………………………………………………………………………22 2.2 Method……………………………………………………………………………22 2.2.1 Construction of flt3l-vp1 fusion gene…………………………………………22 2.2.2 DNA vaccine…………………………………………………………………24 2.2.3 Protein vaccine………………………………………………………………28 2.2.4 Assay of immune response……………………………………………………30 Results…………………………………………………………………………………33 3.1 Construction of flt3l-vp1 fusion gene……………………………………………33 3.1.1 Confirmation of flt3l/pSecTag2/HygroB and vp1/pGEM-T Easy vector………33 3.1.2 Confirmation of flt3l-1/3linker/pGEM-T Easy vector and vp1-1/3linker /pGEM-T Easy vector…………………………………………………………33 3.1.3 Confirmation of flt3l-linker-vp1/pGEM-T Easy vector and flt3l-linker-vp1/ pSecTag/HygroB………………………………………………………………33 3.2 Expression of VP1 protein in E.coli expressing system…………………………34 3.2.1 Confirmation of VP1 expression in small scale………………………………34 3.2.2 The concentration of imidazole in VP1 purification…………………………34 3.3 In vitro functional assay of flt3l-linker-vp1/pSecTag/HygroB……35 3.3.1 Confirmation of expression of flt3l-linker-vp1/pSecTag/HygroB in BHK-21 cell line by Western blotting………………………………………………………35 3.3.2 Confirmation of expression of flt3l-linker-vp1/pSecTag/HygroB in BHK-21 cell line by ELISA…………………………………………………………………35 3.3.3 Analysis of in vitro cultured BM-derived dendritic cells (DC) by flow cytometer………………………………………………………………………35 3.4 Intranasal immunization with plasmids…………………………………………36 3.4.1 Determination of anti-VP1 specific Ab in sera………………………………36 3.4.2 Neutralization of EV71 by sera from DNA-immunized mice…………………37 3.5 Intranasal immunization with proteins…………………………………………37 3.5.1 Determination of anti-VP1 specific Ab in sera………………………………37 3.5.2 Neutralization test of induced antibodies of protein vaccination. ……………39 Discussion…………………………………………………………………………… 40 Figures…………………………………………………………………………………47 Reference………………………………………………………………………………64 Appendix I……………………………………………………………………………71 Appendix II……………………………………………………………………………72 Appendix III…………………………………………………………………………73 | |
dc.language.iso | en | |
dc.title | 利用Flt3L蛋白或Flt3L-VP1重組基因增強腸病毒
疫苗之黏膜免疫反應 | zh_TW |
dc.title | Enhancement of mucosal enterovirus vaccine immune response with Flt3L protein or Flt3L-VP1 fusion gene. | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張鑾英,吳漢忠 | |
dc.subject.keyword | 腸病毒,疫苗, | zh_TW |
dc.subject.keyword | Enterovirus,vaccine, | en |
dc.relation.page | 73 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 口腔生物科學研究所 | zh_TW |
顯示於系所單位: | 口腔生物科學研究所 |
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