Construction Of A Human Fab Phage Antibody Library And The Initial Screening Of Phage Antibody Against IL-4

Background/Objective:Interleukin-4 was discovered in the 1980s, it is a cytokine which has varied functions. It is generated mainly by activated Th2 and mastocyte.IL-4 has crucial role in the pathogenesis of inflammatory,IL-4 can promote the development of inflammatory reaction characterized by TH2 as the eigen cytokine of it, and it have lots of proinflammatory way, however, its comprehensive biological functions make it play a part in the therapy of disease.Especially,it has good results in the treatment of tumors, leukemia and autoimmune diseases.some studies have shown that IL-4 did not only treat many diseases, but also it can lead to the occurrence of some diseases,and It play an important role in the development of some diseases.for example, Asthma,anaphylactic disease,Renal glomerular disease,ovarian cancer and other tumors, cerebral infarction and so on. IL-4 have different changes in the above-mentioned diseases, these changes have great impact in these diseases.therefore, Detecting its changes in the human body is important means in the study of relationship between IL-4 and diseases. Many genetically engineered antibodies have been used in clinical studies,for instance, Rutgers has screenedβ-lardacein antibody from phage antibody library.at the same time, it is also has advantage for diagnosis and treatment of clinical disease if we can create IL-4 antibody through genetic engineering. The main method of the existing to detect IL-4 is ELISA.this method is that coated ELSIA plates by Animals monoclonal antibody to detect IL-4.but the antibody obtained by hybridoma technique can not avoid Heterogeneity problem.this antibody will result in errors to detection.however the antibodies prepared by traditional method have many problems, for example, less, Tedious work, Long cycle, and so on. These problems result in some difficulties in researching IL-4.therefore.preparating the humanized antibody Fast and efficiently has become a solved problem.The phage antibody library technique provides a new idea for preparing the humanized antibody.it make quickly preparing humanized antibody become possible. Phage antibody library from genetically engineered technique.It is that cloned antibody gene into vector. And then showed on the surface of phage.in the end,obtained diversity phage antibody set. this set is phage antibody library, initial, It was expounded by Clackson. Humanized antibody can de screened from phage antibody library. Antibody library technique is a convenient and stable technique by which can screen high-flux humanized antibody.this technique was not through immunity,and bypass hybridoma technique.we can screen specific antibody from antibody library utilizing antigen directly.this technique resolve the problems of hybridoma technique and human body rejection. In our study,We reconstructed a human Fab antibody library on the foundation of origin, with much greater capacity and diversity.To build a platform for preparing IL-4 human antibody.also, To provide necessary premise for antibody expresstion, purification,large-scale preparation and further clinical application research.Methods:In our study, we extracted total RNA from peripheral blood lymphocytes which obtained from healthy donors. The heavy chain Fd and light chain genes were amplified by RT-PCR. and the amplification products were ligated into the phagemid vector pComb3XSS. 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 digested by SacⅠ, XbaⅠ, SpeⅠand Xho I and the phage antibody Fabs or phagemids abstracted from amplified E. coli were amplified by PCR to identify the insertion of light chain or heavy chain Fd genes.then IL-4 was used to screen the original Fab antibody library about five times, and the positive phage antibodies were assayed by Phage-ELISA, Then positive clones were analyzed by DNA sequenced.Results:The capacity of Fab phage antibody library is 2.4×108, It was correct that the insertion of gene fragment was identitied by the digesting of enzymes and PCR. After having been screened about five cycles by IL-4, the original antibody library gained enrichment in 340 times, identified Positive clones by Phage -ELISA have antigen binding activity to IL-4. The DNA sequencing showed that the sequence of Fd fragment had the homology of 91% with y chain and 94% for light chain withλchain of human immunoglobulin.Conclusion:This study successfully constructed a human Fab phage antibody library.Establishing foundation for preparation of various antibodies in the future. Anti-IL-4 Fab antibodies were initially obtained, providing a prerequisite for antibody expression, purification, large-scale preparation in the future, and, preparing for the further research of the relationship between 1L-4 and diseases.