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

• Main Authors: chien-ming hsieh, 謝建銘
• Other Authors: tzong-yuan wu
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/14817629958197455618

碩士 === 中原大學 === 奈米科技碩士學位學程 === 97 === 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.