以噬菌體呈現與單鏈抗體技術發展抗眼鏡蛇蛇毒抗體
指導教授：楊文仁 博士 國立高雄大學生物科技研究所 學生：趙峻毅 國立高雄大學生物科技研究所 摘要 台灣每年約有 500 ~ 1000 人被毒蛇咬傷，注射抗蛇毒血清是目前用以治療被蛇毒咬 傷最有效的方法。目前抗蛇毒血清的製備主要以去毒性蛇毒免疫馬匹後精製而得。然 而，在以馬匹製造抗蛇毒血清過程中，需歷經 3 到 5 個月的去毒性蛇毒針劑注射，常導 致馬匹逐漸消瘦甚至死亡，因此研發新式有效的蛇毒抗體是一項重要課題。本實驗主要 利用噬菌體呈現蛇毒擬抗原決定位免疫小鼠產生抗體以及基因工程產生單鏈變異區片 段抗體(single-chain variable fragment, scFv)兩種方式，開發新的製造抗眼鏡蛇蛇毒抗體 的方法。在噬菌體呈現蛇毒擬抗原決定位方面，將先前篩選出八個代表眼鏡蛇蛇毒不同 抗原決定位的擬抗原噬菌體免疫小鼠後的抗血清混合，進行中和眼鏡蛇蛇毒能力的試 驗，結果發現小鼠存活率為 100％，中和效力為 16 TU (Tanaka unit)。在單鏈變異區片 段抗體製備方面，從具有抗眼鏡蛇蛇毒抗體的小鼠脾臟細胞萃取 mRNA，以聚合酶鏈 連鎖反應擴增出抗體重鏈及輕鏈的變異區基因，建構出單鏈變異區片段抗體基因，將此 抗體基因接合到 pET101 載體後轉形至大腸桿菌中大量表現抗體蛋白。將這些抗體蛋白 混合後，利用動物實驗分析中和蛇毒效果，結果顯示小鼠存活率為 50％，中和效力為 8TU。綜合上述結果顯示，此兩種方式生產之抗體具有中和蛇毒蛋白能力，利用這兩種 策略製造抗體，可以改進傳統製造抗蛇毒血清需耗費大量時間、金錢、人力等缺點，並 提供製造抗蛇毒血清可行的替代路徑。 關鍵字：單鏈抗體、眼鏡蛇、噬菌體呈現技術Development of antibody against Naja naja atra venom using
phage display and single-chain Fv antibody technology
Advisor: Dr. Wen-Jen Yang
Institute of Biotechnology, National University of Kaohsiung Student: Jyun -Yi Jhao
Institute of Biotechnology, National University of Kaohsiung ABSTRACT
It is about 500 ~1000 people bitten by venomous snake in Taiwan every year. The most effective method used to treat the wound by venomous snake is the injection of antivenin. So far, the preparation of antivenin is by injecting horse with detoxified snake venom and then purifies the antiserum. However, it needs to take 3~5 months to immunize the horse for the production of antiserum. It usually causes horse being emaciated gradually and even leads to die. Therefore, it is a crucial topic to develop a novel effective anti-snake venom antibody. Two strategies were used in this study for this purpose. One is the production of antisera using phages displaying the snake venom mimotope on their coat protein as antigen, the other is the production of single chain variable fragment (scFv) antibody by gene engineering. Previously isolated eight phage clones which carrying various mimotopes of Taiwan cobra snake venom were selected to immunize mice to produce antisera. The pooled antisera were used for the cobra snake venom neutralization test. The results showed that 100% survival of the testing mice and equal to 16 TU (Tanaka unit) of neutralizing efficacy. For scFv antibody preparation, mRNA was isolated from the splenocytes of mouse which had high antibody titer against cobra snake venom. The genes encode for variable regions of heavy chain and light chain of antibodies were amplified by PCR to construct the scFv antibody genes. The scFv gene was ligated to pET101 vector and transformed to E. coli for the overexpression of scFv antibody protein. The pooled scFv antibodies were used to analyze the neutralizing efficacy against cobra snake venom in vivo. The results showed that 50% survival of the testing mice and equal to 8 TU (Tanaka unit) of neutralizing efficacy. To sum up, the antibodies produced by above two strategies have the neutralizing capability against snake venom. These strategies could improve the disadvantages including time-consuming, expensive, and labor-intensive of traditional antivenin production and provide alternative pathway for the production of antivenin.
