Construction Of A Camel-derived Naive Nanobody Library And Screening Of Salmonella Typhimurium FlgE Protein Nanobodies

Salmonella Typhimurium is one of the most important pathogenic bacteria causing diarrhea in sheep.Infection with this bacterium commonly causes anorexia,fever(up to 41 ℃)and severe diarrhea in sick sheep,the sick ewes and lambs will show a lot of death,causing significant economic losses to the sheep farming industry,And through contaminated livestock products or contaminated water sources can also threaten human health.Nanobodies(Nbs)are single-domain antibodies that contain only the variable domain of heavy chain of heavy-chain antibody(VHH).The nanobodies have the advantages of small relative molecular mass,high affinity and stability compared with traditional antibodies.They can precisely bind to the surface antigens of diseasecausing bacteria and antagonize the adhesion of bacteria to host cells,which has a wide application in the treatment of bacterial infection-related diseases.The main findings of this paper are as follows:1.Expression and purification of Salmonella typhimurium FlgE.Based on the previous research,the sequence of flgE was obtained from the NCBI database,and the sequence of the protein was clarified by Blast comparison and analysis of signal peptide,transmembrane domain and secondary structure of the protein.pET-28a-flgE,a recombinant plasmid for prokaryotic expression,was synthesized and constructed,and the FlgE protein was expressed and purified using the prokaryotic expression system.2.Construction and identification of a na(?)ve nanobody libraries.A na(?)ve nanobody library with a library capacity of 7.2×1010 cfu was successfully constructed using total RNA was extracted using lymphocytes from peripheral blood and the spleen of unimmunized natural camels;colony PCR validation showed that the library positivity rate was close to 100%;colony DNA sequencing results showed that the constructed na(?)ve library was rich in sequence diversity,It provides a strong platform for subsequent screening of more target-specific Nbs.3.Screening of Salmonella typhimurium FlgE-specific nanobodies.FlgE protein was encapsulated in immunotubes,and after three rounds of panning and phage ELISA identification results showed a total of 26 samples with more than tenfold OD450 determination ratio,and multiple comparison analysis revealed that a total of three different sequences of nanobodies were screened.4.Expression purification and affinity determination of the nanobodies.The screened nanobody sequences were constructed into pADL-23c-Avi-FKPA plasmid,and the VHH-C5 protein with His tag was purified by osmotic shock method.ELISA assay and BLI(Bio-Layer Interferometry,BLI)experiment showed that the recombinant protein VHH-C5 had good affinity with the antigenic protein,the immunofluorescence technique was used to further characterize the binding and agglutination activity of the specific nanobodies to the pathogenic bacteria.5.The eukaryotic yeast expression system was used to successfully express VHH-C5 linked with the IgA sequence.By seamlessly cloning the IgA sequence that can resist protease hydrolysis and the screened VHH-C5 sequence were cloned into the yeast expression vector pPICZαA.The recombinant protein VHH-C5-IgA was successfully expressed in the extracellular of SMD1168 Pichia pastoris.In this study,we constructed a naive library with abundant library capacity,and successfully screened the anti-FlgE flagellin-specific nanobody VHH-C5 by phage display technology.VHH-C5 showed good affinity with the recombinant flagellin and could effectively bind to Salmonella typhimurium and trigger the bacterial agglutination reaction,and further successfully expressed VHH-C5-IgA recombinant protein in the extracellular of Pichia pastoris.This experiment provides ideas and tools for the development of alternative antibiotic therapies for gastrointestinal infections,and also provides an important theoretical basis for the development of orally active antibody formulations.