Construction of scFv Phage Display Library Against Diphenylamine

• Main Authors: Shu-Jian Lin, 林淑娟
• Other Authors: Jung Yie Kao
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/92251249392714967412

碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 92 === Diphenylamine is an antioxidant, having the skeleton structure of non-steroidal anti-inflammatory drugs. In the use of agriculture, it is as a dipping solution to prevent superficial scald in the surface of apple. Diphenylamine also is a fungicide and was used in the pesticides with high concentration. It is an important derivative of BXT, and often used in hair color dye. It is toxic, possible mutagen and teratogen, and harmful in contact with skin. Because diphenylamine is a hapten, in this experiment, we use conjugate reagent to synthesize the bovine serum albumin with diphenylamine together as an immunogen. After immunization, and confirmed with ELISA, we take out the spleen of mouse, and use a series of degenerate primers covering recently known murine immunoglobulin gene family and performing RT-PCR to amplify variable region of heavy chain (VH) and light chain (VL) gene. The amplified VH and VL DNA fragments were assembled to perform single chain fragment of variable region (scFv). The scFv DNA were cloned to the pHEN2 vector, and then transformed to E. coli. We use the technique of phage display system to construct the library of scFv for further selection. The constructed antibody phage display library was confirmed the diversity by BstNI restriction fingerprinting analysis.