Identification And Characterization Of A Novel Nanobody Against Duck Hepatitis A Virus Type 1

Duck virus hepatitis?DVH?caused by duck hepatitis A virus type 1?DHAV-1?is an acute and highly contagious disease affecting young ducklings.The VP1 protein is one of the major structural proteins of DHAV-1 carries critical epitopes responsible for the induction of neutralizing antibodies.Antibody-mediated immunoassays is the most specific and precise assay for detecting DHAV-1.However,traditional monoclonal antibodies used in virus detection need more support costs and they are difficult for massive production.In order to eliminating the drawbacks of conventional antibodies,we aimed to develop nanobodies against DHAV-1.In this study,Camel immunization with live vaccine of DHAV-1 was assessed via ELISA.We have successfully constructed an immune phage display VHHs library against DHAV-1 with the size of 6×10⁶ colonies.After three rounds of bio-panning,a nanobody?Nb?against VP1 protein of DHAV-1,named Nb25,was identified from the immunized phage display library.Nb25 could react with the conserved linear B-cell epitope of¹⁷⁴PAPTST¹⁷⁹in DHAV-1 VP1,which will facilitate the serologic diagnosis of DHAV-1infection.This article is mainly divided into the following three parts:1.The expression of DHAV-1 VP1 proteinA DHAV-1 DNA-launched infectious clone constructed in our previous work was used as templates to amplify VP1 gene.Two restriction sites of EcoR?and Xho?were designed in primers to help to insert VP1 into pET-32a?+?and pGEX-6p-1 prokaryotic expression vector,respectively.The protein was expressed in E.coli Rosetta?DE3?cells after 3 h induction with 1 mM IPTG at 37°C.The recombinant proteins VP1-His and VP1-GST were identified by SDS-PAGE and Western blot.2.Identification and characterization of a novel nanobody against DHAV-1 VP1A male Bactrian camel was immunized subcutaneously six times at biweekly intervals with live vaccine of DHAV-1.Four days after the last immunization,peripheral blood mononuclear cells?PBMCs?were isolated from blood sample.Total RNA was extracted and VHH genes were cloned by nested PCR.The final PCR products were cloned into the phagemid vector pCANTAB 5E and transformed into freshly prepared electro-competent E.coli TG1 cells.The final library was calculated to contain about 6×10⁶ independent clones.The VHH phage library was subjected to bio-panning against captured DHAV-1 VP1-His protein.After three rounds of panning,the phage particles expressing VP1-specific Nbs effectively enriched.Sequence analysis found a unique clone presented in this set of antibody fragments,named Nb25.3.Identification of the liner epitope in DHAV-1 VP1 to Nb25The five overlapping fragments of the DHAV-1 VP1 gene were subcloned into the pGEX-6p-1 expression vector with the help of EcoR?and Xho?restriction sites.The recombinant plasmids were transformed into E.coli Rosetta?DE3?cells to express recombinant proteins.As demonstrated by dot blot assay,the liner epitope recognized by Nb25 was preliminary speculated as ¹⁷²PLPAPTSTTS¹8181 in the DHAV-1 VP1 protein.Subsequently,based on the peptide ¹⁷²PLPAPTSTTS¹⁸¹,a panel of six shortened peptides were generated by deleting amino acids individually,at either the amino or the carboxy terminus in sequence.According to dot blot hybridisation results,the liner epitope recognized by Nb25 was ¹⁷⁴PAPTST¹7979 in the DHAV-1 VP1 protein.The homology alignment results showed that the ¹⁷⁴PAPTST¹7979 was a conserved motif in the VP1 protein of DHAV-1,whereas mutations existed in same sites of DHAV-2 and DHAV-3.