- 學位論文
以桿狀病毒表現系統表現諾羅病毒 VP1 與其融合蛋白之研究
Expression of VP1 of Norovirus and VP1 fusion proteins by baculovirus expressin system
摘要
A 型肉毒桿菌神經毒素 (Botulinum neurotoxins type A；BoNT/A) 為人類常見的食因性毒素，也是生化武器的候選之一。A 型肉毒桿菌神經毒素的蛋白結構分成輕鏈 (light chain；LC) 和重鏈 (heavy chain；HC)，近年的研究指出肉毒桿菌毒素是依靠重鏈和神經元的軸突末端 (Axon terminal) 表面的受體結合，藉由胞吞作用進入神經細胞，此結果顯示此重鏈可以做為有效的疫苗抗原。本實驗將此重鏈蛋白透過連結胜肽與諾羅病毒的衣殼蛋白- VP1 蛋白形成融合蛋白，希望能利用 VP1 上的 P2 subdomain與腸胃黏膜上的組織相容血型抗原 (histo-blood group antigens) 結合而入侵的特性，攜帶肉毒桿菌神經毒素的重鏈蛋白越過腸胃黏膜層，讓免疫系統產生免疫力，以此為概念製作一種口服的肉毒桿菌神經毒素疫苗。本次選用柔性連結胜肽 (GGGGS)2 簡稱 S2，以及剛性連結胜肽 (EAAAK)3 簡稱 A3，來比較兩種不同的連結胜肽對此融合蛋白形成類病毒顆粒是否有影響，並在肉毒桿菌神經毒素的重鏈基因的3’端加上 Histidine tag 方便日後純化。將各個質體完成後，藉由 heat shock 將質體送入 MAX Efficiency® DH10Bac™ 勝任細胞製作重組桿狀病毒質粒 (bacmid)。確認質粒無誤後，轉染至秋行軍蟲卵巢細胞株 (Sf21) 生產重組桿狀病毒。經由終點稀釋法測定病毒力價，以 M.O.I = 0.5 的條件感染秋行軍蟲卵巢細胞株以進行病毒增殖，然後再次以終點稀釋法測定病毒力價。最後以 M.O.I = 1 的條件感染秋行軍蟲卵巢細胞株來生產重組蛋白。透過西方墨點法分析，成功表現了重組 VP1 蛋白之後，再以同樣的條件生產 VP1-linker-BoNT/A HC-His，並使用西方墨點法分析。結果顯示，除了預期的約120 kDa 大小之外，另外出現了約 58~60 kDa 的訊號，目前推測的可能性為部分連結胜肽斷裂的蛋白聚合而成，抑或是只轉譯了部分胺基酸片段，需要透過其他實驗來進一步釐清。未來預計純化重組蛋白，然後以電子顯微鏡觀察重組蛋白能否形成 VLP。
並列摘要
Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most toxic proteins causing the food borne disease and listed among the bio-weapon candidates. The molecule is formed by a heavy chain (HC) and a light chain (LC). The heavy chain C-terminal (HCC) is a receptor binding domain, when it binds to the receptors on axon terminal, the toxin will be endocytosed into nerve cells. In recent studies, the HCC domain has been targeted as a recombinant antigen to generate the neutralizing antibodies. In this study, we fuse the HCC domain with viral protein 1 (VP1) of norovirus (NoV) by fusion linker protein, the VP1 can bind to histo-blood group antigens (HBGAs) on intestinal epithelial cells and infect them, and we expect VP1 could carry HCc to pass through the intestinal epithelial, and be detected by the immune system, as a concept of botulinum neurotoxin oral vaccine. The flexible linker (GGGGS) 2 and rigid linker (EAAAK) 3 were chosen for comparing the effect of the virus like particles assembly, and added a histidine tag at the 3’ end of the HCC for the purification. After completing the plasmid, transform them into MAX Efficiency® DH10Bac™ competent cells to produce bacmid, and then transfect bacmid into sf21 insect cells to produce recombinant baculovirus. The virus titer were analysis by end point dilution assay (EPDA). Amplify the viruses by infecting sf21 with M.O.I = 0.5. Analysis the new viruses titer and infect the sf21 with M.O.I = 1 to produce the recombinant protein. We confirmed the VP1 protein by western blot assay, and used the same condition to produce and analysis the fusion protein VP1-linker-BoNT/A HCC-His. The single that we expect at ~120 kDa had confirmed, but there also showed other single at 58~60 kDa, we suppose those are the protein with the broken linker or it only translate the part of the amino acid sequence. In the future, we will purify the fusion protein, and observe whether the virus like particles are assembled by the electron microscopy.