Construction Of Camelid Single-domain Antibody Library And Perliminayry Biopanning Of Nanobody Against Ractopamine

In the camel immune system,there are antibodies only made up of heavy chains,lacking of light chains naturally,and known as heavy chain antibodies(HCAbs)or nanobodies(Nbs).Compared with the traditional antibody,the nanobody has the unique structure,so that it has a small molecular weight,good solubility and other characteristics,and is widely used in the detection of pathogen,food safety analysis and other fields.Nanobodies have the same antigen binding function equally with full-length antibodies.So it can replace the full-length antibodies in the immunoassay detection of RAC,and establish an immunological detection method with high reliability.Ractopamine(RAC)is a beta-adrenergic agonist that belongs to phenolic amines.RAC can accumulate residues in animal tissue easily,especially the lungs,kidneys,spleen and other organizations.Taking it can make people have headaches,chest tightness and other side effects.At present,the European Union,China and other organizations and countries treat RAC as a prohibited object,forbidden to use it in livestock and poultry.Therefore,the rapid and accurate detection of ractopamine in animal products is of great significance to food safety.The main contents and results are as follows:1.Construction and identification of the camel single domain heavy chain antibody phage display libraryThe RNA was extracted from the peripheral blood lymphocytes of the healthy camels and it had a good integrity.It was reverse transcribed into cDNA.The variable region genes of camel heavy chain antibodies were amplified by PCR.The genes were ligated with the phagemid vector pCANTAB5 E and transformed into E.coli TG1.The recombination rate of the antibody library was about 90%,and the library of 107cfu/mL was constructed.The single colonies were selected randomly and sequenced,which indicated that the antibody library had a good diversity,and could be used for subsequent panning research.2.Synthesis and identification of artificial antigen for ractopamineRAC-OVA artificial antigens were synthesized by using 1,4-butanediol method.RAC-BSA artificial antigens were synthesized by mixed anhydride method.The identifications of artificial antigens were conducted by UV scanning spectrum and SDS-PAGE gel electrophoresis,which indicated the UV absorption peak of the artificial antigens were shifted and the molecular weight of the conjugate was larger than the carrier protein.It was proved that the artificial antigens of RAC-OVA and RAC-BSA were coupled successfully.3.Screening and identification of recombinant phage for anti-ractopamine nanobodyRecombinant phagemids expressing anti-RAC specific nanobody were screened from the phage display antibody library by artificial antigen RAC-BSA.The positive phage was amplified and purified to obtain the crude protein of soluble recombinant antibody.The crude extract of nanobody was further detected by ELISA,which indicated that it has antigen binding ability.In this study,we have successfully constructed a camelid native single-domain heavy chain phage display antibody library,and screened anti-ractopamine phage particles,which laid the foundation for the expression of anti-ractopamine nanobodies and the immunological detection of RAC.The construction of the native phage display antibody library laid the foundation for the subsequent panning of other nanobodies for specific antigens.