以桿狀病毒表現系統生產A型肉毒桿菌毒素做為疫苗可行性的分析

Recombinant botulinum neurotoxin A production by baculovirus expression system and evaluated for vaccine development

謝建銘(chien-ming hsieh)

指導教授 ： 吳宗遠

中原大學/理學院/奈米科技碩士學位學程/碩士(2009年)

https://doi.org/10.6840/cycu200901190

摘要

A型肉毒桿菌神經毒素為肉毒桿菌所生產的外毒素，其作用位置在於神經突觸及神經肌肉聯會處，功能為阻斷神經傳導物質釋放，造成肌肉處於鬆弛狀態，且只需極低的劑量即可造成長時間的肌肉鬆弛狀態。如被有心人士利用作為生化武器，可造成大範圍傷害，因此，開發疫苗為重要的工作。
我們利用桿狀病毒表現系統表現A型肉毒桿菌神經毒素重鏈全長 （BONT/A HF）、C端 （BONT/A HC） 及綠螢光融合A型肉毒桿菌神經毒素重鏈C端（EGFP‧BONT/A HC），並且在在其C端末端融合6個組胺酸，以IMAC套組純化前述蛋白。
以m.o.i = 1的病毒感染條件，並在第四天收取細胞，使用IMAC套組純化BoNT/A HC重組蛋白，其中產量約為287μg，純度約為90%，並以抗體進行西方點墨法分析。BoNT/A HF於本表現系統中無法量產，推測其原因可能為AT在整段基因中佔了81%，昆蟲細胞對於如此高AT比例的基因無法順利轉譯。EGFP‧BONT/A HC融合蛋白能在本表現系統表現，產出具有螢光特性的融合蛋白，觀察GFP是否能增強BoNT/A HC的表現量，並應用在標定神經細胞的實驗。
我們所生產的BoNT/A HC重組蛋白委請國防醫學大學預防醫學研究所趙德江博士協助動物免疫的實驗，成功引發老鼠的免疫反應，使老鼠產生針對A型肉毒桿菌神經毒素的中和性抗體，而使免疫後的老鼠能抵抗肉毒桿菌神經毒素。

關鍵字

疫苗 ； 桿狀病毒表現系統 ； A型肉毒桿菌毒素

並列摘要

Botulinum neurotoxin type A (BoNT/A) is produced by Clostridium botulinum. It can break neurotransmitter release at neuron-muscle junction and synapse.
Baculovirus expression vector system (BEVS) is used to to express botulinum neurotoxin type A heavy chain full length (BoNT/A HF), C terminal (BoNT/A HC) and EGFP fused with BoNT/A HC protein with the condition of their C terminal fused with six His. The protein can be purified by using IMAC.
We use condition m.o.i = 1 and harvest cellular sample at fourth day. Then we purify BoNT/A HC by using IMAC. The quantity of output is about 2.5% and the quality near 90%. Next, we also analyze BoNT/A HC by western blotting analysis. However, the BoNT/A HF-6h does not work out. The possible reason is AT occupies 80% of the sequence while insect’s cells cannot proceed clone or translation in such high proportion of AT. The fusion protein produced by fusing green fluorescence with BoNT/A HC-6h is both with the characteristics of fluorescence. And we also observe the EGFP can enhance BoNT/A HC production or not and using this fusion protein apply to neuron marker.
We request Dr. Chiao to process immune assay experiment. In immune assay experiment on mice, recombinant BoNT/A HC-6h does successfully induce the mice’s immune reaction and protects the mice from invasion of toxins.

並列關鍵字

baculovirus expression system ； vaccine ； botulinum neurotoxin A

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