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研究生:王阡樺
論文名稱:A型流行性感冒病毒M2核酸疫苗增強免疫效果之研究
論文名稱(外文):Enhancement of the antibody response after administration of Influenza A virus M2 DNA vaccine
指導教授:李君男李君男引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫事技術學研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:62
中文關鍵詞:A型流行性感冒病毒M2蛋白質核酸疫苗增強免疫
外文關鍵詞:influenza A virusM2 proteinDNA vaccineenhancement of immune response
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流行性感冒病毒,簡稱流感病毒,會造成急性上呼吸道感染,藉由空氣迅速的傳播,在世界各地常會有週期性的大流行。流行性感冒病毒在免疫力較弱的老人或小孩及一些免疫失調的病人會引起較嚴重的症狀,如肺炎或是心肺衰竭等。本研究主要欲探討如何藉由肌肉注射流感病毒DNA疫苗,提高宿主對此疾病的免疫力。
以肌肉注射DNA疫苗的主要轉染(transfection)的對象為肌肉細胞,由於肌肉細胞並非血液類細胞,故沒有第二型組織相容性抗原(MHC class II)的表現,也不會表現共刺激分子,故在核酸免疫中主要扮演的角色為表現外源蛋白質。
本研究以pcDNA3為載體,為了能表現可溶性蛋白質,而將A型流感病毒的M2蛋白質之穿膜部位第25-55胺基酸去除(M2d25-55,pMd),為了能加強抗原的表現,在其N端結合上Staphylococcus Aureus蛋白質A(protein A)的訊息序列(pSMd),以期能加強抗原的表現。此外,由於抗原呈現細胞的細胞膜上有許多的Fc受器,因此在M2d25-55之 C端結合人類γ免疫球蛋白Fc部位(pSMdFc),預期可以更有效的被抗原呈現細胞吞噬,並呈現在第二型組織相容性抗原上,進而引起較佳的抗體反應。另外,本研究以Th2細胞激素IL5為佐劑,期以促使已活化的成熟B細胞增加抗原特異性sIgA的表現,以加強對流感病毒感染的保護效果。
將上述質體轉染293T細胞發現,無論M2d25-55蛋白質及另兩種建構的蛋白質SMd 及 SMdFc均無法在細胞質中偵測其表現。以大腸桿菌表現的重組蛋白質MBP-MdFc與小鼠巨嗜細胞株(P388D1)進行結合試驗,發現重組蛋白質的Fc部位確實可以透過與Fc受器的結合而較有效率的將Md蛋白質導向巨嗜細胞。在動物實驗方面,施打pSMdFc或合併注射pIL-5相較於接受pMd 或是pSMd的BALB/c與C57L/B6小鼠,確實能有效的提升血清中M2蛋白質特異性抗體的產生。
本論文利用融合Fc部位來增強核酸疫苗所引起的免疫反應,期待未來能應用於增強免疫性較弱的抗原核酸疫苗上。

Influenza virus can cause acute respiratory tract infection in humans. Epidemics of varying severity occur annually. It has caused severe morbidity or even mortality, especially in the elderly and children whose immune responses are weakened or not well established. Therefore, the development of a suitable vaccine is urgently needed.
Intramuscular injection is most often applied in DNA vaccination. Myocytes are the major targets to be transfected by this method. Because myocytes are nucleated cells with few MHC class I and no MHC class II on the cell membrane, it is considered to be an “antigen production factory”.
In this study, we cloned the gene encoding the influenza A virus M2 protein deleted at transmembrane region, residues 25-55 (pMd). In order to enhance the expression by the host cell, signal peptide of Staphylococcus aureus protein A was fused to the N-terminus of the M2d25-55 (pSMd). Meanwhile, human IgG1 Fc fragment was fused to the C-terminus of M2d25-55 (pSMdFc) to target the antigen expressed to the antigen presenting cells. Moreover, Th2 cytokine, IL-5, was cloned and used as the adjuvant to augment the M2 specific IgA response.
293T cell was transfected by each of the three plasmids and found that no M2 protein could be detected in the cytoplasm. Using E.coli as the expression host to express MBP-MdFc, we demonstrated that Md antigen was indeed targeted to the murine macrophage cell line, P388D1, via the interaction of the fused Fc and Fc receptor on P388D1 cell. In the in vivo experiment, influenza A virus M2 specific antibody was detected in the mice immunized with pSMdFc alone or with pIL-5, however, none of the mice immunized with pMd or pSMd alone or with pIL-5 could be detected to be M2 specific antibody positive.
In this study, we had successfully demonstrated the effectiveness of Fc fusion in the enhancement of M2 antibody response after administration of DNA vaccine. This showed the Fc fusion with the potential as an alternative approach to enhance the immune response differing from the applications of other traditional adjuvants.

Content
英文摘要…………… 1
中文摘要…………… 2
Introduction……… 3
Material and Methods …………………… 12
1.Mice……………………………… 12
2.Antibody………………………… 12
3. Cell line………………… 12
4. PCR reaction and plasmid construction…… 12
5. DNA sequencing…………………………… 13
5-1. Cycle sequencing………………………………… 13
5-2. DNA precipitation by ethanol……………………… 14
5-3. Sequence analysis and alignment…………………… 14
6. Construction of DNA vaccine……………… 14
6-1. Enzyme digestion…………………………………… 14
6-2. Ligation and transformation………………………… 15
6-3. Colony PCR………………………………………… 15
6-4.Plasmid preparation………………………………… 15
7. In vitro transfection……………… 16
7-1. Cell transfection and plasmid gene expression… 16
7-2. RAN extraction and RT-PCR……………………… 16
7-3. Time course of plasmid gene expression and
immunoprecipitation………………………………… 17
7-4. SDS-PAGE and western blot………………………… 17
8.Fc targeting assay…………………………… 18
8-1. Construction of recombinant M2Fc protein………… 18
8-2. Small-scale expression of MBP-M2Fc
recombinant protein………………………………… 19
8-3. Large-scale expression of MBP-M2Fc protein……… 19
8-4. Fc binding assay……………………………………… 19
8-5. Fc-mediated internalization assay…………………… 20
9.Measurement of murine IL-5…………………… 20
10.Animal vaccination and evaluation of
antibody responses……………………… 21
10-1. Animal vaccination by intramuscular
and intranasal method…………………………… 21
10-2. Evaluate the antibody response by ELISA………… 21
11.Neutralization test………………………………………… 22
12.Viral challenge…………………………………………… 22
Result…………………………………………………… 23
1.Contruction of DNA vaccine pSMd and pSMdFc
and adjuvant pIL-5…………………………………………… 23
2.RNA expression level of each construct……………… 23
3.Protein expression of each construct in vitro……… 23
4.Expression of MBP-M2Fc recombinant protein…………… 24
5.Fc-mediated binding and internalization of MBP-M2Fc… 24
6.Measurement of murine IL-5 by ELISA…………………… 25
7.M2 antibody response in immunized mice………………… 25
8.Neutralization ability of M2 antibody………………… 26
Discussion………………………………………………… 27
Figure………………………………………………………… 34
Appendix…………………………………………………………… 51
Reference………………………………………………………… 53

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