Construction And Identification Of Camelid Natural Nanobody Phage Display Library And Screening For Anti-GDH Nanobody

Objective To construct a natural nanobody phage display library as a nanobody platform for screening different antigens,and identify the display library.Using GDH antigen to screen and obtain single domain antibody variable region gene(VHH)of the camel targeting GDH,then make it prokaryotic expression and purificated it.Methods 1.We Isolated the total RNA from the camel by using Trizol,and used Oligo dT to synthesize the cDNA.The camel heavy chain variable region sequence was obtained from the NCBI database,and based on the constant region sequences,we designed the nested PCR primers.The genes of variable domain of heavy chain(VHH)were amplified by nested PCR,and then were ligated with the vector pCANTAB5 e by digestive enzyme and DNA ligase.Next the recombinant vector cloned into TG1 to construct the phage display library,and make the analysis and identification for the library.2.We efficiently expressed GDH antigen by prokaryotic expression system and purified it by the Ni column system.We used GDH antigen to coat the ELISA plate and screened the phage display library.After three rounds of "adsorption-elution-amplification" panning process,we obtained phagemids containing the target gene,which can specifically bind to the GDH antigen.And then identified the monoclonal by phage-ELISA.3.We constructed the VHH sequence,which obtained target gene,into the PET30 a expression vector,and efficiently expressed anti-GDH recombinant Nanobody by prokaryotic expression system.At the same time,we can use the Ni column system to obtain the purified antibodies after purification,and the antibodies were verified by SDS-PAGE and Western Blot analysis.Conclusion 1.We successfully constructed the camelid natural nanobody phage display library.After identifying the quality of the library,the positive rate of the phage display library is 95%,the amino acid homology of nine randomly colonies is 66.17%,and it has a good variety after MEGA analyzing.In addition,they also belong to domain antibody variable region gene after BLAST comparison.2.After three rounds of panning screening for the phage library with GDH antigen as the target,the phage was effectively enriched and the recovery rate was gradually increased.After identification by ELISA,two anti-GDH Nanobody sequences were finally obtained,named anti-GDH-01 and anti-GDH-02.3.After cloning the VHH sequence into PET30 a expression vector for prokaryotic expression,SDS-PAGE analysis showed that the recombinant protein was expressed as inclusion body and the size was about 45 kDa.The recombinant protein was purified by using His tag and analyzed by Western Blot.In western blot,the nanobody recombinant protein was used as the primary antibody,and the anti-human IgG/HRP was used as the secondary antibody for Western Blot analysis.And finally,the result showed that the recombinant nanobody could specifically bind to GDH antigen,and the specificity was good.Finally,we obtained 8mg antiGDH purified Nanobody.