A RANDOM SINGLE CHAIN ANTIBODY LIBRARY

THE OVERALL GOAL OF THIS PROJECT IS TO CONSTRUCT A SINGLE CHAIN ANTIBODY (SC-AB) EXPRESSION LIBRARY. DEGENERATE PCR PRIMERS WILL BE USED TO AMPLIFY ANTIBODY HEAVY AND LIGHT CHAIN VARIABLE REGIONS. HEAVY AND LIGHT CHAINS WILL BE RECOMBINED IN VITRO USING PCR AND LINKED WITHA 42 BP SPACER ARM TO MAKE FUNCTIONAL SC-AB. THE SC-AB LIBRARY WILL BE EXPRESSED IN A MODIFIEDLAMBDA VECTOR (LAMBDA LOX) UNDER THE CONTROL OF A BACTERIOPHAGE T7 PROMOTER WHICH ALLOWS IPTG INDUCED PRODUCTION OF TARGET PROTEINS IN AMOUNTS EXCEEDING 50% OF TOTAL CELL PROTEIN. ANOTHER FEATURE OF THE SYSTEM IS EASY CONVERSION OF THE BASE LAMBDA VECTOR TO A PLASMID FOR EXPRESSION.THE SC-AB WILL CONTAIN AN OLIGO-HISTIDINE STRETCH TO SERVE AS AN AFFINITY TAG FOR RAPID PURIFICATION BY METAL CHELATION CHROMATOGRAPHY. THE CAPACITY OF THE VECTOR TO RECEIVE AND EXPRESS AN ANTIBODY WILL BE TESTED USING AN ANTI-C5A MONOCLONAL. IN THE FINAL PHASE OF THE WORK, A SC-AB RECOMBINATORIAL LIBRARY WILL BE CONSTRUCTED FROM PERIPHERAL BLOOD B CELLS OF INDIVIDUALS IMMUNIZED WITH A HEMOPHILUS INFLUENZA TYPE B CONJUGATE VACCINE. BOTH CONTROL ANTIBODY TARGETS (C5A AND H. FLU) HAVE THERAPEUTIC POTENTIAL. THE TECHNOLOGY WILL PERMIT THE GENERATION OF SINGLE CHAIN ANTIGEN BINDING MOLECULES WITHOUT RESORT TO MONOCLONALS.