| Background/Objective:Amylin is a peptide hormone produced and stored in the β-islet cells of pancreas along with insulin. They will be coreleased by certain proportion from β cells when stimulated by glucose and non-sugar hormones. As a kind of pleiotropic hormone, amylin plays multiple roles in the substance and energy metabolism in our body. Recent research reveals that, IAPP oligomers, formed by the abnormal metabolism of amylin, is early mesostate in the formation of amyloidogenic peptides. involved in the destruction of the cell lipid bilayers of β cells and leading the apoptosis of (3cells. Therefore, the relationship between amylin and type2diabetes mellitus is paid much attention by more and more researchers. However, the exact biological functions of amylin are still unclear. Nevertheless, it’s difficult for us to detect the hormone due to the small molecular and the low level in vivo. And so far, the methods of detecting the hormone are complex and antibodies, prepared from hybridoma approach and immune animal, are lack of stability and specificity. Therefore, how to prepare high purity amylin antibody by an easy and fast way is the key point for further research.The phage display technology opened a new way to prepare human antibodies, made a possibility to pan human antibodies fragments, and solved the difficult problem of preparing antibodies. It is a stable and simple technology to prepare high quality human antibodies by means of genetic engineering. It can directly pan specific antibodies against antigens from antibody libraries through phage display technology. In our study, we construct a human Fab antibody library, with much greater capacity and diversity, to provide a platform for amylin human antibody expression, purification and large scale preparation in further study. Meanwhile, it also provides a feasible method to further clinical application.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 PCR with variable regions5" and3’ primers of heavy and light chain, 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 VCSM13helper phage to yield recombinant phage antibody Fabs. The phagemids abstracted from amplified E.coli were digested by Sac Ⅰ. Xba Ⅰ. Spe Ⅰ and 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. Amylin was used to pan the original Fab antibody library, and the positive phage antibodies were assayed by Phage-ELISA analysis. Then positive clones were analyzed by DNA sequenced.Results:By combination of light chain and heavy chain genes, an antibody library containing0.8×108clones was obtained and the insertion rate of target fragment was70%. Both the digesting 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 by amylin, the original antibody library gained enrichment in22times, which indicated that anti-amylin Fab antibodies were enriched remarkably. Phage-ELISA was performed to determine if the supernatant that contained the amylin Fab antibodies had specificity and binding reactivity towards amylin. The DNA sequencing showed that the sequence of Fd fragment had the homology of88%with y chain and93%for light chain with λ chain of human immunoglobulin.Conclusion:This research successfully constructed a human Fab phage antibody library, providing a technology platform for preparation of various antibodies in the future. Anti-amylin Fab antibodies were successfully obtained, which prepared to express. purify the antibodies and study the physiological and pathological functions of amylin in future research. |
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