Development of antibody against Naja naja atra venom using phage display and single-chain Fv antibody technology

• Main Authors: Jyun - Yi Jhao, 趙峻毅
• Other Authors: Wen-Jen Yang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/05052536340982686964

碩士 === 國立高雄大學 === 生物科技研究所 === 98 === 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.