Expression Of ORF2 Protein Of Novel Goose Astrovirus In Baculovirus And Its Application In Antibody Detection

Goose astrovirus(GAstV)belongs to the genus avian astrovirus and its genome includes three open reading frames(ORF1a,ORF1b,and ORF2).The ORF2 gene encodes the capsid protein of astrovirus which is associated with the pathogenicity of the virus.In recent years,infectious diseases caused by the novel GAstV have caused outbreaks in major farming areas in eastern China,characterized by the occurrence of urate deposits on the surface of internal organs and joint cavities.GAstV infection shows high pathogenicity in young geese,with a lethality rate of up to 50%,which seriously threatened the development of the goose industry in China and caused huge economic losses.To date,commercial serologic diagnostic technique for the detection of novel specific antibodies against GAstV is not available.The capsid protein encoded by the ORF2 gene is the major structural protein of GAstV and can induce neutralizing antibodies and immune response.In this study,we aimed to use the capsid protein encoded by the ORF2 gene of GAstV as the detection target,and establish an indirect ELISA for the detection of antibodies against GAstV by efficient expression of capsid protein using a baculovirus expression system.1.Expression of ORF2 protein of GAstV in baculovirus and its purificationThe capsid protein encoded by the ORF2 gene of the GD strain of GAstV was first cloned into the pFast-Bac-HTA baculovirus vector to construct a recombinant shuttle vector plasmid.The plasmid was transfected into Sf9 insect cells to obtain a recombinant baculovirus expressing ORF2 gene,designated as rBv-ORF2.Indirect immunofluorescence assay(IFA)using monoclonal antibody against ORF2 showed that specific bright green fluorescence was observed in Sf9 cells infected with rBvORF2.Western blot analysis showed that a ORF2 specific protein band that match the expected size was found at 78 kDa.ORF2 protein was purified from the supernatant of cell lysate infected with recombinant baculovirus using a Ni+column and the concentration of purified ORF2 protein was 0.33 mg/ml.SDS-PAGE and Western blot indicated that the purified ORF2 protein was approximately 78 kDa in size and showed good immunogenicity and reactivity,which laid the foundation for the development of immunological approachs for GAstV and the study on ORF2 gene function.2.Development of indirect ELISA for detection of GAstV antibodyUsing purified ORF2 protein as a specific coating antigen,an indirect ELISA method for detecting antibodies to GAstV was established,designated as ORF2_ELISA.ELISA reaction conditions including coating protein concentration,blocking solution,primary antibody serum dilution and reaction time were optimized by square titration,and the Cut-off value of the ELISA was determined to be 0.4(S/P value).Specificity and repeatability tests showed that the established indirect ELISA only reacted with GAstV positive serum,but not with the positive serum of other goose susceptible viruses,and repeatability coefficients of variation was below 10%,with good repeatability;In the sensitivity analysis test,the sensitivity of ELISA could reach 4 times of IFA;The clinical sample test results showed that the concordance rate of this ELISA method with the IFA test was 89.8%.The above results indicate that the ORF2 protein of GAstV was successfully expressed in baculoviruses in this study,and an indirect ELISA method with good specificity,sensitivity,and repeatability was established.This study provides an accurate,fast,and economical detection method for clinical infection detection and seroepidemiological investigation of GAstV,providing technical support for the development of clinically applicable detection kits.