2013年，新型禽流感H7N9病毒在中國大陸沿海城鎮爆發。H7N9病毒屬於低致病性禽流感病毒，但在感染人類後會產生急性呼吸道窘迫症並導致死亡，死亡率已高達37%，至今仍無可對抗H7N9病毒的疫苗上市。目前有關H7N9病毒疫苗的研究仍以肌肉注射的免疫方式為發展主軸，但H7N9病毒是經呼吸道感染，因此，本研究主要是以鼻噴劑的策略，發展H7N9重組血凝素黏膜疫苗。
此研究中使用中國倉鼠卵巢細胞(CHO/dhFr-)與草地夜蛾蟲(Sf9)細胞生產重組H7HA蛋白，製備成黏膜疫苗後免疫小鼠，結果顯示Sf9細胞表現的蛋白有較高的IgG與IgA效價、HI效價與NT效價，可以引起較強的免疫反應；而CHO細胞表現的蛋白本身不具引發免疫反應的能力，必須添加佐劑後才能引起小鼠體內的免疫反應。接著，使用Sf9細胞所生產的蛋白搭配不同的佐劑，研究何種佐劑最適合H7N9黏膜疫苗。結果指出CpG與PELC混合而成的佐劑，能在小鼠黏膜組織與血清中有較佳的IgG和IgA效價，其次是第二型b型忌熱型腸毒素(LTIIb)混合PELC的組合。最大的發現是Sf9所生產的H7HA蛋白在不需添加佐劑的情況下，依舊可以引起小鼠黏膜組織與血清中IgG和IgA效價，且僅略低於搭配細菌鞭毛蛋白(FliC)或是第二型b型忌熱型腸毒素(LTIIb)的黏膜疫苗。結果顯示Sf9細胞表現的H7HA重組蛋白本身可能是一個具發展潛力的候選黏膜疫苗。

The novel avian-origin influenza A/H7N9 virus cause outbreak in China coastal towns in 2013. The H7N9 virus is belongs to low pathogenic avian influenza virus, resulting in acute respiratory distress syndrome in human which infected with virus and human death. The mortality rate of H7N9 is about 37%. Until June 2015, there is no available vaccine against H7N9 virus infection. Most of the vaccines are administered intramuscularly, whereas human get infected with the H7N9 virus through respiratory tract. Previous research in 2014 shows that after two doses immunization of recombinant baculovirus subunit mucosal vaccine, the mice can survive in H7N9 virus challenge experiment. These results show that it is feasible to develop mucosal vaccines. Thus, in this study we developed the rH7HA mucosal vaccine using intranasal immunization strategy. In this study I used Chinese hamster ovary cells (CHO / dhFr-) and Spodoptera insect (Sf9) cells to produce recombinant H7HA protein and used these protein as mucosal vaccines to immune mice. The result showed that the protein produced by Sf9 cells (rH7HA-Sf9) induced high IgG, IgA, HI and NT titers in mice serum and caused strong immune response. But proteins expressed by CHO cells (rH7HA-CHO) without adjuvants did not have the ability to elicit an immune response in mice. Next, I used rH7HA-Sf9 formulated with different adjuvants as mucosal vaccines to find the best rH7HA-Sf9/adjuvant combination for H7N9 mucosal vaccine development. The result indicate that compared with other adjuvants, CpG mixed with PELC as adjuvant formulated with rH7HA-Sf9 induce the highest IgG and IgA titers in mice mucosal tissues and serum. Using type IIb heat-labile toxin B subunits (LTIIb) mixed with PELC as adjuvant also can induce high IgG and IgA titers. The interesting finding is that rH7HA-Sf9 proteins without adjuvant can cause higher IgG, IgA titers in mice mucosal tissue and serum than those induced by protein formulated with R848, and slightly lower than those induced by protein formulated with flagellum (FliC) or type IIb heat-labile toxin B subunits. The rH7HA-Sf9 proteins may be a candidate for H7N9 mucosal vaccine development.

摘要 II
Abstract II
致謝 IV
一、簡介 1
1.1. A型流感病毒 1
1.2. H7N9新型禽流感病毒 1
1.3. H7N9疫苗的發展 2
1.4.黏膜組織的免疫反應 4
1.5.黏膜疫苗佐劑開發 6
1.6.類鐸受體新型黏膜佐劑開發 6
二、研究目標 9
三、材料與方法 10
3.1.小鼠來源 10
3.2.重組蛋白(rHA)序列 10
3.3.細胞株 10
3.4.製造重組桿狀病毒 11
3.5.使用Sf9細胞表現並純化蛋白 12
3.6.利用CHO細胞表現並純化蛋白 12
3.7.SDS膠體電泳法(SDS-PAGE) 13
3.8.膠體染色法(Coomassie Blue Staining) 13
3.9.蛋白濃度測定 13
3.10.西方墨點法(Western Blotting) 14
3.11.Endo H與PNGase F與血球凝集素(HA)的作用 14
3.12.胎球蛋白結合試驗(Fetuin-binding assay) 15
3.13.胎球蛋白結合性抑制實驗(fetuin-binding inhibition assay) 15
3.14.血球凝集反應試驗 16
3.15.黏膜疫苗免疫的組別 16
3.16.黏膜疫苗製備 17
3.17.黏膜疫苗免疫 17
3.18.小鼠免疫樣品採集 17
3.19.酵素結合免疫吸附分析(Enzyme-Linked Immunoasorbent Assay) 18
3.20.血球凝集反應抑制試驗(Hemagglutinin inhibition assay) 19
3.21.抗體中和能力試驗(Neutralization assay) 19
3.22.T細胞之細胞激素分析 20
四、結果 21
4.1.重組H7HA蛋白的表現與特徵分析 21
4.2.不同蛋白免疫小鼠組別後血清中對H7HA專一性抗體的產生 23
4.3.血清中抗體的中和能力 24
4.4.不同黏膜佐劑組別血清中對H7HA專一性抗體的產生 25
4.5. 不同黏膜佐劑組別支氣管及肺部沖洗液中對H7HA專一性抗體的產生 26
4.6.不同黏膜佐劑組別細胞激素的產生 28
五、討論 30
六、參考文獻 35
七、圖檔 44
Table 1 黏膜疫苗接種組別 44
Table 2 黏膜疫苗免疫組別 45
Fig.1 CHO/dhFr-細胞與Sf9昆蟲細胞表現的重組H7HA蛋白特徵 46
Fig.2 血球凝集素抑制試驗與胎球蛋白結合實驗 47
Fig.3 不同免疫組別血清中對H7HA專一性抗體的產生 48
Fig.4 不同免疫組別產生的血凝素抑制性抗體與抗體中和性試驗 49
Fig.5 重組H7HA蛋白與胎球蛋白結合能力抑制實驗 50
Fig.6 不同免疫組別血清中對H7HA專一性抗體的產生 51
Fig.7 不同免疫組別支氣管及肺部沖洗液中對H7HA專一性抗體的產生 52
Fig.8 不同組別血清中抗體對血凝素功能的抑制性 53
Fig.9重組H7HA蛋白與胎球蛋白結合能力抑制實驗 54
Fig.10 不同組別脾臟細胞與周邊淋巴結細胞中細胞激素的產生 56
Fig.S1 Th17細胞活化的訊息傳遞路徑 57

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