表現肉毒桿菌素之蛋白質區塊以應用於臨床檢測抗肉毒桿菌素之抗體

Abstract

肉毒桿菌 (Clostridium botulinum) 可生產八種具不同特性的外毒素 (type A, B, C1, C2, D, E, F, and G)，並作用於神經肌肉會合處阻礙乙醯膽鹼釋放，以中斷神經訊號傳導，造成肌肉癱瘓。雖然肉毒桿菌素 (botulinum neurotoxin) 是自然界中毒性最強的物質之一，但是調整使用量後，則可以應用於治療斜視、面肌痙攣與某些類型的語言障礙等。此外，由於其低副作用與高耐受性，更使得注射肉毒桿菌素成為流行的醫美項目，然而長時間接受肉毒桿菌素注射，可能會因患者產生中和性抗體而降低效果甚至失效，因此有必要發展一種評估患者對肉毒桿菌素抗性的工具。現行用於醫療與醫美主要是A型肉毒桿菌素，本研究於大腸桿菌中分別表現A型肉毒桿菌素的受體結合區 (receptor-binding domain)、易位區 (translocation domain) 與肽酶區 (peptidase domain)，再以管柱層析法進行純化，以建立可以偵測抗A型肉毒桿菌素抗體之酵素聯結免疫吸附分析法。本研究成果有助於檢測患者之抗藥性生成情形，並藉此調整治療方法及避免無效醫療。

Clostridium botulinum produces eight antigenically distinctive exotoxins (type A, B, C1, C2, D, E, F, and G), which can block the release of acetylcholine and the neurotransmission at the neuromuscular junction for causing muscle paralysis. Though botulinum neurotoxin (BTX) is one of the most poisonous biological substances known, it has also been used as therapies for strabismus, hemifacial spasm, some types of language disorder, and the cosmetic surgery after dose adjustment. The few side effects and well tolerability made injection of neurotoxin a popular cosmetological application. However, in long-term BTX treatment, loss of therapeutic efficacy may occur due to induction of the neutralizing antibody, which causes non-responsiveness to treatment. Therefore, it’s necessary to develop a method for dermatologists to evaluate the efficacy of BTX before injection in patients. The BTX type A has been widely used for medical and cosmetological application. In this study, the receptor-binding domain, translocation domain and peptidase domain of BTX type A were expressed in E. coli, and purified by column chromatography. The protein domains of BTX were applied in establishing an enzyme-linked immunosorbent assay for detecting the anti-neurotoxin antibody. The present study developed a useful tool for monitoring the formation of the anti-neurotoxin antibody in patients and can be considered as a diagnostic index for preventing futile medical cares.