Construction, Identification And Screening Of Humanized Fab Fragment Phage Antibody Library Against Schistosoma Japonica

Antibody technology has a history of antiserum of multiclone antibody and monoclonal antibody while phage display antibody library technology is most significant development for the present. The production of traditional monoclonal antibody was limited to very laborious and time-consuming processes involving animal immunization schemes and/or hybridoma generation. While phage display antibody library has made it easy and a breakthrough in production of antibody. The technology uses filamentous phage as vehicle which extrinsic gene can be inserted into. The fused phage with extrinsic gene has ability of amplification and expressing extrinsic antibody. After several rounds of binding-elution-enrichment procedure , the target phages are enriched which have specific binding ability .The technology phage solves problem of low effect in hybridoma system and opens up extensive perspective for producing humans monoclone antibodies because it does not need cell hybridization and avoids instability of hybridoma cell line. Therefore phage antibody display library is considered to be the third generation for antibody development after hybridoma antibody. Though the period of phage display antibody librarys application is short , it shows great potentiality and extensive applied perspective in cancers diagnosis and therapy,epitope analysis,drug design and etc. The main objective for the experiment is to construct a humanized Fab fragment phage antibody library against Schistosoma japonica using phage display antibody library technique and to obtain humanized antibody against Schistosoma japonica. First, The blend of patients' sera that have resistance against Schistosoma japonica was used as antibody to immunoscreened cDNA Library constructed. Positive clones were sequencing. As a result, SPI(serine protease inhibitor) which have a complete open reading frame was chosen to be amplified by PCR,cloned , expressed and purified for phage display library screeening. Second, Light chains and heavy Fd fragments genes of IgG were obtained by RT-PCR from peripheral blood lymphocyte of adult who have resistance to Schistosoma japonica. Then they were cloned into PComb3 vector; following transformed into bacillus coli Xl1-blue and superinfected with helper phage to finish construction of humanized Fab fragment phage antibody library whose size was 6. 2×1011and volume was 1.2×107. The library was identified with restriction enzyme digestion and sequence analysis, DOT-ELISA, SDS-PAGE and etc. Results show that a humanized Fab fragment phage antibody library against Schistosoma japonica was constructed successfully and specific antibody against Schistosoma japonica SPI was obtained by ELISA methods from screening the phage antibody library after several rounds of binding elution enrichment procedure.The results of the experiment show that a humanized Fab fragment phage antibody library against Schistosoma japonica was constructed successfully with peripheral blood lymphocyte of adult, which makes a solid foundation for further studying immunologic,characteristics and functions of these antibodies.