Preparation Of Nanobodies Against A Truncated Protein Of Avian HEV ORF2 And Development Of A Competitive ELISA

Avian Hepatitis E virus(a HEV)is the main pathogen of Big liver and spleen disease(BLSD)and Hepatitis-splenomegaly syndrome(HSS)in chickens,which mainly causes a decrease in egg production and an increase in mortality.The diseased chickens usually have abdominal congestion,ovarian degeneration,liver fat or amyloidosis,which seriously affects the development of the breeding industry.For the serological detection of avian HEV,there is no recognized commercial test kit at present,and the established indirect enzyme-linked immunosorbent assay(ELISA)has high requirements on the purity of the coated antigen,which often has has an influence on background value level.In this study,a competitive ELISA was developed to detect serum antibodies against avian hepatitis E virus(HEV).For the c ELISA,the coating antigen is a truncated protein containing C-terminal268-amino acid region(aa339-606)of ORF2 from an avian HEV strain isolated in China and contains the main epitope of the virus particle,was named Ca268 in this study.Nanobody,also known as the variable region of the heavy chain(VHH),is the variable region of the camel-specific Ig G(Ig G2 and Ig G3).It has the advantages of small molecular weight,stable structure,good solubility,strong specificity,and ability to recognize special epitopes.In this study,the Ca268 protein was used to immunize Bactrian camels,and phage display technology was used to obtain specific nanobodies against Ca268,and one of the strains was selected to establish a competitive ELISA.The results are as follows:1.The p ET21b-Ω-Ca268 recombinant plasmid constructed in the early stage of the laboratory was transformed into E.coli Rosetta(DE3)p Lys S,after induction via IPTG,the Ca268 protein with high purity was harvested in the form of inclusion body.The protein was refolded by gradient concentration of urea,and the final yield was about 50 mg/L.2.Through 5 immunizations of Bactrian camels,the peripheral blood lymphocytes were separated and the VHH gene was amplified,then connected it into the p MECS vector and transformed into TG1 competent cells,a nanobody library with a library capacity of7×10~9 was obtained.Using phage display technology for 3 rounds of panning,6 strains of specific nanobodies against Ca268 protein were obtained.3.The Ca HEV-Nb49 nanobody gene was inserted the p EGFP-N1-RANbodies vector and was transfected into HEK293T cells for eukaryotic expression of the Ca HEV-Nb49-HRP fusion,which was used as competitive ELISA detection reagent.After exploring and optimizing conditions,a competitive ELISA for serological detection of avian HEV was finally established.4.The Ca268 protein was continued to be truncated and expressed to find epitopes binding with the Ca HEV-Nb49-HRP fusion,and a short peptide was synthesized for verification.The position of the epitope was determined to be aa593-604.Comparing the homology of the different genotypes of the avian HEV ORF2 protein in this region,the mutation of the different amino acids will not affect the recognition of the nanobody.In summary,this study screened and prepared specific nanobodies against Ca268protein,and successfully expressed nanobody-HRP fusion,which was used as detection reagent of c ELISA to detect serum antibodies against avian HEV.Compared with the established indirect ELISA(i ELISA),c ELISA reduces the use of HRP-labeled secondary antibodies,shortens the detection time,and simplifies the operation process.At the same time,this method has a high consistency rate with i ELISA and Western Blot,and it can theoretically detect serum antibodies all genotypes of avian HEV.In view of the above advantages,the c ELSIA established in this study can be widely used for serological investigation of avian HEV infection in chicken flocks.