Construction Of Human Naive Phage Display Antibody Library And Primary Screening Of The Fab Antibodies

Objective: To study the construction of human naive phage display antibody library and primary screening of the Fab antibodies of malignant lymphoma patients with the method of genetic engineering. Methods: With lymphoprep,peripheral blood lymphocytes were isolated from 200ml blood,which was obtained from lymphoma patient. The heavy chain Fd and light chain cDNA synthesized from the total RNA of lymphocytes were amplified by PCR with variable regions 5'and 3'primers of heavy and light chain,and the amplification products were ligated into the phagemid vector pComb3,then the ligated sample was transformed into competent E.coli XL1-Blue by Electroporation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as SacⅠ, XbaⅠ, SpeⅠand XhoⅠand bother the phage antibody Fabs or phagemids abstracted from amplified E.coli were amplifiedby PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment. Human albumin panning to the orginal Fab antibody library.Result:The quantity of total RNA and cDNA were qualified.By ombination of light chain and heavy chain genes,an antibody library containing 6.6×106clones was obtained,and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.After having been panned respectively by three kind of antigen proteins,the original antibody library gained enrichment in different degree.Conclusion: Utilizing the technology of phage surface display,special antibody can be gained from the human naive Fab phage display library,which can be used as a new therapy for tumors.