Human Antibody Library Construction By Phage Display

Objective:Construst human genetic engineering antibody library on phage to screen human single chain antibody of anti-HBs.Methods:1. To separate B-lymphocytes from healthy volunteers' blood, and extract total RNA from B-lymphocytes2. To reverse transcript mRNA into cDNA with Oligo(dT) primer according to the charater of mRNA3.To design a set of oligonucleotide primers to amplify the cDNA of immunogloblulin heavy and light chain variable domains (Vh and Vl) by PCR4.To amplify the linker gene by PCR to link Vh and Vl5.To link Vh and Vl genes by a 15-amino acid linker--(Gly4Ser)3 toform a single chain Fv (ScFv)6.To Clone the ScFv gene into phagemid vector pCANTAB5E and tranform E.Coli TGI, with the help of the phage M13K07, so antibody fragments are expressed on the surface of phage7.To collect the human antibody library to identify and screen our interesting antibody with ELISAResults:1. The mRNA from B-lymphocytes was reverse transcripted into cDNA2. We successfully amplified Vh and Vl gene fragment ,and could see the expected bands from gel.3. We amplified the linker gene about 100bp by PCR4. We cloned the vector pCANTAB5E/ScFv and formed many positive clones on plate with antibiotin ampicilin5. We collected the human phage antibody library to identify and screen the phage with anti-HBs antibody fragmentConclusionWe expect to screen ScFv of anti-HBs by constructing a human antibody library, thus can avoid human anti-mouse antibody (HAMA) reaction. Because the ScFv is not so long as complete immnogolbulin, and hasn't the constant regions, we can connect the ScFv gene and rIFN gene to express together as a promising gene therapy method against HBV infection. Moreover, the genetic engineering antibody satify the need of many patients in order to prevent the infection of HBV between mother and baby. In a word, anti-HBs ScFv antibody has clinically extensive applied prospects.