Construction Of Human Na(?)ve Phage Display Antibody Library And Primary Screening Of The Fab Antibodies Against Gp96

Background/Objective: It has long been known that the cellular heat shock protein gp96 derived from cancer cells or tissues can elicit cancer-specific immunity. Recent studies have indicated that gp96 molecules, as a chaperon, participate in the presentation of the associated peptides to T cells through histocompatibility complex class I -restricted antigen presentation pathway. On the basis of these features, gp96 can be used for cancer immunotherapy. However, this very promising application will require the rapid purification of gp96 molecules from a limited amount of sample material at a very high purity. The conventional procedure to isolate gp96 molecules requires ConA-Sepharose column purification, which result in contamination of gp96 with ConA due to bleeding from the column. Because ConA is a T-cell mitogen, it might harmfully interfere with the vaccinee's immune system. Using phage antibody library can avoid from the trouble depicted previously. In addition, the anti-gp96 Fab antibodies speed up the purification process and allow the processing of small tumor samples.Phage surface display technique, which has become an increasingly important tool in biotechnology, provides a new way for antibody development. In this project, we constructed a naive human Fab fragment phage display library, provide a platform for human antibody preparation and a basis to new therapy for the malignant tumors.Methods: With lymphoprep, peripheral blood lymphocytes were isolated from 800 ml blood, which was obtained from four healthy blood donors. 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 XL 1-Blue by Electroporation.The transformed cells were infected with VCSM13 helper phage toyield recombinant phage antibody Fabs. The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac I , Xba I , Spe I and Xho I and bother the phage antibody Fabs or phagemids abstracted from amplified E.coli were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment. Human albumin, interleukin-2 and gp96 were utilized as antigens to conduct respectively three rounds of panning to the original Fab antibody library.Results: The quantity of total RNA and cDNA were qualified. By combination of light chain and heavy chain genes, an antibody library containing 6.6x 106 clones 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 kinds 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.