Preparation Of Anti-Human DR5Single Chain Variable Fragment Antibody By Phage Surface Display Technique

BackgrandDR5is one of tumor necrosis factor （TNF）-related apoptosis inducing ligand （TRAIL）’s receptors.Thisapoptotic effect is mediated by interaction of TRAIL and its receptors. Our laboratory have prepared thehybridoma named YM366EC, which can secrete anti-DR5antibody. It can cause human anti-mouseantibody reaction because it is a murine monoclonal antibody. With the development of genetic engineeringtechnology, we have prepared the humanized and full human antibody can be prepared. Through thistechnology, we not only overcomed the disadvantage of the murine antibody, but also preserved specificityand homogeneity of the murine antibody, The preparation technology of gene engineering antibodyincludes the following three aspects: chimeric antibody, CDR transplantation antibody, antibody preparedby display technique and transgenic technique. The HAMA reaction has been overcomed because thedisplay technique can prepared full human antibody. So, phage display technique is the optimal methods tothe prepartion of full human antibody..ObjectiveTo establish a phage antibody library by phage display technology and screen he full human antibodywhich anti-DR5.MethodsVH and VL of human antibody were obtained by RT-PCR. Peptide gene （Gly₄Ser）3.connectedVH/VL into a single chain antibody scFv through overlap extension PCR. Then, scFV gene was clonedinto the expression vector pcantab-5E using restriction enzyme SfiI and Not â… . They were electroporat into escherichia coli X-Blue. The primary phage antibody library was established after phage infection.. Toidentify the insertion rate of the antibody library, we use the bacterium liquid PCR. We sequenced thepositive clones and analized the diversity of the phage antibody library. The anti-DR5positive clonesscFv were picked up from the primary phage antibody library after four rounds of enriched by ELISAmethod.ResultsWe have successfully constructed a natural phage antibody library of1.01×107, which have a gooddiversity. Finelly, We find that12clones is the same in30clones, and4clones are highly homologous toanti-TNF-α antibody.ConclusionsWe have screened out several strains which highly expess scFv segment of the full human anti-DR5antibody,and make a good basis for the preparation of target protein. We have established a natural fullhuman antibody library technology platform, which lay the foundation for future screening other fullhuman antibody which anti toxins, viruses, bacteria and obtain specific full human antibody drugs.