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研究生:林岱昀
論文名稱:應用PTD-J-X免疫系統複合熱休克蛋白70及樹突細胞於疫苗之研究
論文名稱(外文):The usage of PTD-J-X immunization system with heat shock protein 70 and dendritic cells on the vaccination
指導教授:張立雪張立雪引用關係莊景凱
學位類別:碩士
校院名稱:國立新竹教育大學
系所名稱:應用科學系碩士班
學門:自然科學學門
學類:其他自然科學學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:81
中文關鍵詞:PTD-J 區塊熱休克蛋白70樹突細胞口蹄疫病毒人類端粒反轉錄酶
外文關鍵詞:PTD-J domainHsp 70Dendritic cellsFMDVhTERT
相關次數:
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中文摘要

在以E. coli表現重組蛋白質的系統中,當重組蛋白質表現量高時,幾乎皆為不可溶的未摺疊蛋白質。藉由和Hsp40 J domain形成融合蛋白質J-X polypeptide,則可藉與負責伴引之Hsp70 Nucleotide binding domain (NBD)間的特異結合,將未摺疊的蛋白質送到Hsp70之基質摺疊,提高摺疊成正確構形之蛋白質的機會。J-X融合重組蛋白質再加上可穿越細胞膜的protein transduction domain (PTD),就成為PTD-J-X重組蛋白質,可穿越細胞膜成為細胞內抗原,誘發Th1/Tc細胞之細胞免疫反應,而未穿越細胞膜的細胞外PTD-J-X重組蛋白質亦可誘發Th2/B細胞之體液免疫反應。
本論文嘗試利用口蹄疫病毒(FMDV) 蛋白鞘VP1結構蛋白 的Th epitope加B epitope (FMDVepi)來誘發體液免疫反應,以及人類端粒反轉錄酶(hTERT) 的Th epitope加Tc epitope (hTERTepi)來誘發細胞免疫反應;同時,也嘗試連結Hsp70 (特指誘發型Hsp72)會和 Hsp40 J domain親和以及Hsp70可激活樹突細胞 (Dendritic cells , DC)的特性,測試Hsp70是否可以在PTD-J-X epitopes免疫機制上扮演佐劑的角色。
體液免疫反應以FMDVepi、PTD-J-FMDVepi、PTD-J-FMDVepi/mHsp70 (murine Hsp70) 及FMDVepi/mHsp70做抗原免疫BALB/c小鼠後,藉由點漬法分析、西方轉漬法分析、免疫螢光染色來偵測對抗FMDVepi及FMDV VP1全長蛋白的免疫反應。結果顯示注射PTD-J-FMDVepi/mHsp70複合體之小鼠的特異性抗體價數明顯較高。細胞免疫反應則以DCs、PTD-J-hTERTepi/DCs、PTD-J-hTERTepi /mHsp70/ DCs經腹腔注射免疫小鼠後,進行淋巴細胞增殖分析、IFNγ及TNFα酵素免疫點分析法和腫瘤抑制試驗。結果顯示增殖能力及IFNγ的分泌皆以PTD-J-hTERTepi/mHsp70/DCs最佳,而TNFα的分泌相對的差異較小。另外,不論先施打或後施打PTD-J-hTERTepi/DCs均可明顯抑制腫瘤的生長。綜合上述結果指出,PTD-J-X免疫系統複合Hsp70及樹突細胞確實能誘發顯著的免疫反應,進而大幅增強疫苗效能。

Abstract

By using the specific interactions between the J-domain of Hsp40 which holds unfolded proteins and the nucleotide binding domain of Hsp70 which folds the denatured proteins into a native conformation, we have proposed a PTD-J-X recombinant protein model and demonstrated that it could be expressed in large amount with higher solubility in comparison with the trxA-X counterpart in E. coli because of its convenience to attach to the Hsp70 chaperone via the J-domain. Besides the protein folding activity, Hsp70 also acts as a specialized carrier allowing the processed antigen peptides (epitopes) coupled to MHC-I molecular intracellularly and as an endogenous danger signal in the immune system. Here we want to demonstrate the X-antigen presenting characteristics of the PTD-J-X recombinant protein. The membrane penetrating peptide, PTD (protein transduction domain), of the PTD-J-X recombinant protein can carry the covalently-conjugated J-X polypeptide passing through the cell membrane. That is to say, the PTD-J-X recombinant protein is expected to act both as extra-cellular and intra-cellular antigens to elicit the humoral and cellular immunity, respectively.
FMDVepi composing of a Th epitope and B epitopes of the FMDV VP1 capsid protein was used as the X-antigen for the humoral immunity model and hTERTepi which was constructed by two sets of overlapped Th/Tc epitopes of the hTERT protein was chosen as the X-antigen for the cellular immunity model. The recombinant mouse Hsp70 (the inducible form Hsp72) was tested whether it could be utilized as a protein adjuvant to enhance the X-antigen presentation for the PTD-J-X recombinant protein. Four set of FMDVepi compositions, FMDVepi, PTD-J-FMDVepi, PTD-J-FMDVepi/mHsp70 and FMDVepi/mHsp70, mixing with Freund’s adjuvant were injected s.c. to BALB/c mice to test whether the antibodies elicited can recognize the full length VP1 protein or not. All of the three sera from the PTD-J-FMDVepi/mHsp70 set can detect the denatured form and native form of VP1 demonstrating by western blotting and immunofluorescent, respectively.
Since dendritic cells (DCs) were found as the most potent antigen presenting cells, in vitro expanded bone marrow derived DCs were utilized in the PTD-J-hTERTepi induced cellular immunity experiments to improve the sensitivity. Three sets of compositions, DCs, PTD-J-hTERTepi/DCs and PTD-J-hTERTepi/mHsp70/DCs, were injected i.p. into BALB/c mice and activated lymphocytes in spleen were analyzed by lymphocyte proliferation assay and INFγ- and TNF-ELISPOT assays. In comparison to the DCs set results, PTD-J-hTERTepi/DCs could stimulate immune responses and Hsp70 could enhance the responses significantly. Therefore, we tried to investigate whether PTD-J-hTERTepi/DCs could be used as vaccine or therapy in cancer treatment. Either pre-treatment or post-treatment of BALB/c mice with PTD-J-hTERTepi/DCs could reduce the CT26 (hTERT) tumor sizes. Taken together, the PTD-J-X model plays a powerful role in the immune system, both in the humoral and cellular branches, and the Hsp70 acts as an omnipotent adjuvant for the PTD-J-X antigen.

中文摘要 I
英文摘要 II
誌謝 IV
目錄 V
圖目錄 VIII
第一章 緒論

1 1-1. 免疫系統, 體液性免疫及細胞性免疫 1
1-1-1. 先天性免疫 1
1-1-2. 應變性免疫 1
1-2. 樹突細胞 2
1-2-1. 樹突細胞系及其發育 2
1-2-2. 體外備製樹突細胞 4
1-3. 熱休克蛋白70 (Hsp70)與Hsp 40作用於蛋白質摺疊 4
1-4. Hsp70應用於免疫調節: 細胞內Hsp70 5
1-5. Hsp70應用於免疫調節: 細胞外 Hsp70 5
1-5-1. Membrane associated Hsp70 6
1-5-2. Extracellular Hsp70 6
1-5-3. Exosomal Hsp70 7
1-6. PTD-J-X 系統 7
1-7. 口蹄疫病毒 (Foot-and-Mouth Disease Virus, FMDV) 8
1-7-1. 口蹄疫暴發層出不窮 8
1-7-2. FMDV與口蹄疫 9
1-7-3. 口蹄疫疫苗 9
1-8. 端粒反轉錄酶(Telomerase reverse transcriptase, TERT) 10
1-8-1. 端粒與端粒酶 10
1-8-2. TERT與癌症治療 12

第二章 實驗材料與方法
2-1. mHsp70基因選殖 13
2-1-1. 聚合酶鏈反應 (Polymerase chain reaction, PCR) 13
2-1-2. 基因選殖 14
2-2. 設計FMDV、hTERT抗原基 14
2-2-1. FMDV抗原基 14
2-2-2. hTERT抗原基 16
2-3. FMDV、hTERT抗原基cDNA合成及Assembly PCR 18
2-3-1. cDNA合成 18
2-3-2. Assembly PCR 19
2-4. 表現及純化重組蛋白 21
2-4-1 建構重組蛋白表現質體 21
2-4-2. 重組蛋白表現與可溶性測定 21
2-4-2. Ni-NTA-agarose管柱純化 22
2-5.構築pCX-FMDV VP1-Flag2及pCX-hTERT-Flag2-IREShyg 質體 22
2-6. 建立CT26 (hTERT) 細胞株 23
2-6-1. CT26細胞培養 23
2-6-2. DNA轉染 (transfection) 23
2-6-3. 篩選穩定表現 hTERT之細胞株 23
2-7. 製備骨髓衍生的樹突細胞 24
2-7-1. 分離骨髓細胞 24
2-7-2. 培養骨髓衍生的樹突細胞 24
2-8. 體液性免疫試驗 24
2-8-1. FMDVepi重組蛋白點漬分析 25
2-8-2. FMDV VP1 重組蛋白西方點漬法分析 26
2-8-3. 免疫螢光染色 26
2-9. 細胞性免疫試驗 26
2-9-1. 淋巴細胞增殖分析 27
2-9-2. 酵素免疫點分析 ( ELISPOT) 27
2-9-3. 腫瘤抑制 28
2-10. 統計分析 29

第三章 結果與討論
3-1. 抗原基設計分析 30
3-1-1. FMDV抗原基 30
3-1-2. hTERT抗原基 30
3-2. 重組蛋白質的大量表現及純化 32
3-2-1. mHsp70 重組蛋白的大量表現及純化 32
3-2-2. PTD-J-FMDVepi與FMDVepi的大量表現及純化 33
3-2-3. PTD-J-hTERTepi的大量表現及純化 35
3-3. CT26 (hTERT) 細胞株之hTERT表現量 36
3-4. 體外培養骨髓衍生的樹突細胞之特性 38
3-5. 體液性免疫試驗-以FMDV為例 40
3-5-1. 誘發對抗FMDVepi特異抗體之能力 40
3-5-2. 誘發對抗denatured形式FMDV VP1之抗體 43
3-5-3. 誘發對抗native形式FMDV VP1之抗體 44
3-6. 細胞性免疫試驗-以hTERT為例 45
3-6-1. PTD-J-hTERTepi誘發針對hTERT特異免疫反應 45
3-6-2. PTD-J-hTERTepi 誘發抗癌活性分析 47

第四章 結論與未來展望 49
第五章 參考文獻 50
附錄 56

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