Construction And Preliminary Application Of Nanobody Synthesis Library

BackgroundIn order to obtain high-affinity and high-specificity antibodies,the preparation of antibodies has undergone a long history of development.Nowadays,antibodies are prepared mainly by hybridoma techniques and genetic engineering techniques.The use of scFv libraries is the most common method of engineering antibody.However,due to the limitations of their size and affinity,antibodies derived from this method often show poor stability.The study has found that there is an antibody that naturally depletes the light chain in the peripheral blood of alpaca.The antibody contains only one heavy chain variable region(VHH)and two conventional CH2 and CH3 regions,but unlike the artificially modified ones.The chain antibody fragments(scFv)are easily sticking to each other and even clump together.More importantly,this antibody has the same structural stability as the diabodies and binding activity to antigens.It is the smallest unit known to bind target antigens and is called a Nanobody(Nbs).Nanobodies extracted from Camelidae are more stable than conventional antibodies due to their smaller size and other biochemical characteristics.Nanobodies have been proven to be great value as therapeutic molecules and clinical diagnostic tools.The conventional procedure for obtaining Nbs is to immunize camels with antigens,but when the antigens are highly toxic,pathogenic or non-immunogenic,the camels cannot be immunized,thus counteracting certain limitations.Synthetic nanobody libraries have no restrictions on the antigens to be screened and are more convenient to use.There are six complementarity determining regions(CDRs)of conventional monoclonal antibodies.However,it has been found that the nanobody derived from Camelidae has only three natural complementarity determining regions.It does not affect the function of the Nanobody,in which the CDR3 of the Nanobody has the greatest diversity and is most important for the function of the Nanobody.The phage antibody library technology is a new technology and method formed by the combination of antibody gene library technology and phage display technology,and has great potential in the field of biological sciences.A phage antibody is a single-stranded phage that expresses an antibody molecule on the surface of a phage.This surface expression is achieved by the fusion protein forming a fusion protein with a single-stranded phage coat protein(PIII or PVIII).As a result,antibody genes are present in the phage DNA.Existence,and the expression of antibody molecules on the surface,can conveniently use the principle of antigen-antibody specific binding to screen out the required antibodies through multiple rounds of antigen adsorption-elution-amplification.In this study,we selected the camel VHH scaffold as a scaffold and randomized the amino acid sequence of the CDR3,constructed and synthesized a diverse nanobody synthesis library.Then,we sceen antibodies by Taq DNA polymerase and immunoglobulin antibodies(IgG)as screening antigens.PurposesA Nanobody synthesis library was constructed.On the platform of the nanobody synthesis library,phage display technology was used to screen affinity antibodies against Taq DNA polymerase and immunoglobulin antibodies.Methods1.Based on the camel conserved single domain antibody fragment(VHH)framework,diversity was introduced into the complementarity determining region 3(CDR3)by randomization of synthetic oligonucleotides.This constructed a large and diverse nanobody synthesis library.2.Prokaryotic expression and purification of Taq DNA polymerase and detection of its enzymatic activity.3.Screening affinity antibodies of Taq DNA polymerase and immunoglobulin antibody from the library using phage display technology.Results1.Successfully constructed a Nanobody synthesis library with a storage capacity of 10~8.2.Successfully constructed and purified the prokaryotic expressed Taq DNA polymerase,which can be used in the PCR reaction and successfully catalyze the PCR reaction.3.Affinity antibodies against Taq DNA polymerase and immunoglobulin antibodies were successfully screened out.ConclusionThe Nanobody synthesis library in this experiment is rich in diversity and has application value,providing a good platform for the subsequent screening of other antibodies.