臺灣博碩士論文加值系統

目前所使用的流行性感冒病毒疫苗因為主要是以去活性的病毒所組成，但歸因於病毒表面蛋白質HA及NA的抗原變異性太大，需要每年進行評估及調整。為了改善這個問題，本實驗室選擇了另一個保守性較高的病毒表面蛋白質M2來做研究，觀察分析M2重組蛋白質之特異的免疫反應，探討以M2重組蛋白質做為疫苗材料的潛力。
本實驗室之前的研究發現，利用帶有M2蛋白質全長基因的質體pcDNA3（pMfull）免疫小鼠，可以誘發高量的特異性抗體。此外，更構築了M2的重組蛋白質基因MdFc，在去除掉M2蛋白質穿膜部分基因（Md），並以五個胺基酸（G-G-G-G-S）之連結子相接之M2基因序列的5’端接上Staphylococcus aureus A的secreting signal胺基酸片段的DNA，並在其3’端接上人類免疫球蛋白IgG的Fc片段，構築成pMdFc。以之免疫小鼠，再佐以含有小鼠IL-5 基因（pIL-5）的質體，結果發現也可誘發高量的特異性抗體。
本研究主要接續以上的研究，增加重組蛋白質MdFc之連結子長度，構築了pMdFc系列的質體。此外，亦將Fc片段接在Md蛋白質基因的5’端以增加其表現及分泌，構築了pFcMd系列的質體。與pMfull比較，並觀察質體在細胞中的表現以及免疫小鼠後所產生的免疫反應。
結果發現轉染細胞之後有mRNA產生，但是卻無法直接在細胞中偵測到重組蛋白質的存在。免疫小鼠後，收集小鼠血清，以純化過的GST-M2重組蛋白質當作抗原，進行ELISA試驗偵測小鼠血清中M2特異性抗體的變化。結果顯示所構築的質體均能誘發M2特異性抗體的產生，產生的抗體量最高為pMfull，其次為pMdFc 系列，再來才是pFcMd 系列。此外，佐以注射pIL-5則能夠刺激較高抗體量的產生，分析IgG抗體亞型發現IgG1有顯著的升高。進一步觀察免疫小鼠的以ELISPOT試驗偵測細胞性免疫反應，小鼠的脾臟細胞經過前處理後加入已含有INF-γ特異性抗體之96孔為量滴定盤中，以GST-M2當作刺激物，再以avidine標記之INF-γ抗體偵測分泌INF-γ之脾臟細胞，最後以AEC呈色，計數產生隻斑點數。發現構築的質體均能引起小鼠的細胞性免疫反應，由強至弱依序為pMfull、pMdFc系列、pFcMd系列。與抗體反應之結果相符合。但是以A/PR/8/34病毒株來測試則發現產生的抗體並無中和病毒的能力。
根據實驗結果，未來研究的方向或許可利用pMfull佐以適當的佐劑來當做流行性感冒病毒DNA疫苗的材料，進一步探討M2蛋白質基因做為DNA疫苗成份的潛力。

Influenza virus causes acute respiratory infection in humans. Epidemics occur annually. It can cause severe mortality especially in the elderly and children. The influenza vaccine being used now is composed of two inactivated Influenza A virus and one inactivated Influenza B virus. Due to the antigenic variation of viral surface proteins, hemagglutinin (HA) and nuraminidase (NA), the vaccine has to be evaluated and adjusted every year. In order to improve the situation, this study choses another virus surface protein, M2, which is highly conserved, to study. Trying to observe and analyze the specific immune response of recombinant M2 protein and to evaluate the potential of the plasmid DNA expressing recombinant M2 protein as influenza DNA vaccine.
The previous studies of our lab showed that administrating mice with plasmid containing the sequence of M2 gene（pcDNA-Mfull, pMfull）could induce antibody response. In addition, our lab also constructed the plasmid pcDNA3-MdFc（pMdFc）. The Md portion was formed by deleting the transmembrne domain of M2 protein and a linker contains 5 amino acid（G-G-G-G-S）was added between extracellular and intracellular domains. The secreting signal peptide of Staphylococcus aureus protein A was added to the N-terminus of Md and human IgG Fc fragment was added to the C-terminus of Md to form MdFc. The muce immunized with pMdFc and plasmid containing mouse IL-5 sequence（pIL-5）as the adjuvant were also induced high antibody response.
In this study, we continued previous studies and tried to enhance the expression and the immune responses of recombinant M2 protein. The different lengths of linker between Md and Fc was added, to construct a series of plasmids. On the other hand, the human IgG Fc fragment was moved to the N-terminus of Md, linkers with different length was added between Fc and Md to construct pFcMd group. Then we compared these two groups of plasmids with pMfull after tranfecting cell and administrating mice.
The mRNA of recombinant M2 protein could be detected after transfecting cells, however, there was no detectable recombinant M2 protein. The animal experiments showed that pMfull, pMdFc group, and pFcMd group all could induce high antibody responses, and the titers from high to low were in the order of pMfull, pMdFc group, and pFcMd group. Furthermore, immunization mice with pIL-5 as the adjuvant resulted in higher antibody responses than those without pIL-5. Analyzing the IgG subtype of induced antibody showed that pIL-5 not only could increase the amount of total antibodies but also significantly raise the amount of IgG1 antibodies. The result of ELISPOT, which detected the cellular immune responses, corresponded well to that of antibody response.
According to the study above, though there was still much left to be studied about M2 protein in the future, pMfull with appropriate adjuvant may be a potential DNA vaccine for influenza.

中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………3
緒論………………………………………………………………………5
實驗材料與方法………………………………………………………..11
I. 材料來源…………………………………..………………………11
1-1病毒來源………………………………………………………..11
1-2 DNA載體... ………………………………………….…………11
1-3 A型流行性感冒病毒M2蛋白質之單株抗體……………..….11
II. 質體DNA的製備…………..………………………...…………...11
2-1構築不同的M2蛋白質表現質體……………….…..………...12
2-2質體之製備……………………………………..………..……12
III. 細胞實驗……………………………………………..………….15
3-1轉染MDCK細胞…………………………………………......15
3-2偵測目標基因mRNA…………………………………………16
3-3質體在轉染MDCK細胞上的表現…………………………...17
3-4穩定表現M2蛋白質之MDCK細胞株……………………….19
3-5 Fc片段之APC導向實驗……………………………….……..19
IV. 動物實驗………………………………………………………...20
4-1肌肉注射與採血………………………………………………..20
4-2酵素連結免疫吸附實驗（ELISA）……………………………21
4-3 ELISPOT………………………………………………………..21
4-4中和試驗………………………………………………………..22
結果……………………………………………………………………..24
質體的製備…………………………………………………………..24
偵測目標基因在轉染MDCK細胞中的mRNA表現………………24
質體在轉染MDCK細胞上的表現…………………………………….25
SDS-PAGE與西方墨點法………………………………………...25
螢光免疫染色法…………………………………………………..25
穩定表現M2蛋白值得MDCK細胞株………………………….25
以M2 DNA質體免疫小鼠…………………………………………..26
以酵素連結免疫吸附實驗偵測小鼠接種疫苗後M2特異性抗體力價的改變…………………………………………………………..27
偵測小鼠抗體IgG亞型的分布…………………………………...27
中和反應…………………………………………………………..28
ELISPOT…………………………………………………………..28
Fc片段之APC導向實驗……………………………………………29
討論……………………………………………………………………..30
圖………………………………………………………………………..33
表………………………………………………………………………..48
附錄一…………………………………………………………………..49
附錄二…………………………………………………………………..50
附錄三…………………………………………………………………..51
附錄四…………………………………………………………………..52
附錄五…………………………………………………………………..53
參考資料………………………………………………………………..54

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