Molecular cloning and Expression of Anti-aflatoxin B1 Monoclonal Antibody Fragment

• Main Authors: Chin,I-Shan, 金益善
• Other Authors: Tseng,Tsung-Che;Lin,Chan-Pin
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/90924925374403064667

碩士 === 國立臺灣大學 === 植物病蟲害學系 === 81 === A molecular cloning approach has been conducted to express anti- aflatoxin B1 (anti- AFB1) monoclonal antibody (MAb) in Escherichia coli. Complementary DNA (cDNA) was synthesized from poly (A)+ RNA isolated from hybridoma 1F7 that secrets specific anti- AFB1 MAb. Using specific primer pairs complementary to heavy chain and light chain genes of mouse antibody (IgG), subsets of cDNA responsible for coding the heavy and light chains of immunoglobulin were selectively amplified by polymerase chain reaction (PCR). A recombination of modified bacteriophage λ expression vectors and the PCR amplified DNA was applied in the generation of a combinatorial library in E. coli. Two IgG Fab clones (1F7001 and 1F7802) specific for AFB1 were selected. Using an indirect comptitive ELISA, Fab from clone 1F7802 showed the binding specificity for AFB1 with no cross-reactivity to AFM1, the same as that of the MAb from hybridoma 1F7. In the affinity test with indirect ELISA using AFB1- BSA as coating antigen, the minimal detectable concentration of AFB1-BSA for the Fab from both clones were as low as 0.01 ppm , the same as that of the Mab from hybridoma 1F7. Periplasmic protein of both clones was analyzed by non- reducing or reducing SDS-PAGE together with immunoblotting. A full-length of Fab fragment (50 Kd) was observed. in the non- reducing conidition , while a Fd fragment (28 Kd) was observed when reducing condition was applied. For recombinant plasmid of clone 1F7802 ,DNA sequences responsible for the varilable regions of the Fd fragment and κ light chain shared 94 and 90 percent homology with those of the known heavy and κ light chain of mouse IgG, respectively.