Preparation Of The Neutralizing Monoclonal Antibodies And Epitopes Identification Of The S1 Glycoprotein Of Avian Infectious Bronchitis Virus Sczy3

Avian infectious bronchitis (IB) caused by the avain infectious bronchitis virus (IBV), is an highly contagious respiratory infectious disease. It can infect chickens at all ages and replicate in many tissues, causing respiratory symptom, diarrhea, and decline of egg production and quality, etc, resulting in huge economic losses in poultry industry. In this study, the monoclonal antibodies against the S1 glycoprotein of Sczy3 which classified into QX genotype were prepared and used to identify the antigenic epitopes of the S1 glycoprotein. It was helpful to further understanding the molecular structure and the antigenic structure of the S1 glycoprotein, and providing the theoretical bases to develop the epitope vaccines and diagnostic reagents.First, the bioinformatics analysis was taken to the S1 glycoprotein of Sczy3, the result demonstrated that the secondary structure of S1 glycoprotein were composed mainly of β-strain and β-turn containing little irregular coil regions, having many amphipathic a-helix and (3-strain regions, flexible regions and surface probability regions, and well immunogenicity. In this study, the strong antigenicity region located in 81aa-422aa of S1 glycoprotein was chosen for prokaryotic expression. The nucleotide sequences were amplified and cloned into pET-32a (+) vector to generate recombinant plasmid pET-32-S1. The positive recombinant plasmid pET-32-S1 was transformed into the competent cell E.coli BL21 (DE3). The result of SDS-PAGE showed that recombinant protein about 57.5 kDa was successfully expressed though IPTG induction. After optimizing of the concentrate of IPTG and the inducement hour, the expression protein His-S1 was abundantly expressed. Then the expression protein was purified with HisTrap FF crude by affinity chromatography, and the purification effect was more than 90%. The recombinant protein His-S1 was reacted with BABL/c mice hyperimmune serum against IBV by Western blot analysis. The result showed that the protein can react positively with mice hyperimmune serum against IBV. The recombinant protein His-S1 could be used to screen the mAbs against S1 protein of Sczy3.IBV Sczy3 strain was further purified using differential velocity centrifugation, and then the BABL/c mice were immunized with purified Sczy3 particles for 4 times. The ELISA method was built with the BALB/c mice hyperimmune serum and negative serum. The spleen cells of the immune mice and SP2/0 cells were fused with PEG 1500 three times. The fusion cells were firstly detected by whole viral antigen-based indirect ELISA, the positive hybridomas were secondly screened by the recombinant protein His-Sl-based indirect ELISA. The hybridomas were further tested by recombinant S1-based ELISA and Western blot. Two hybridoas against Sczy3 S1 protein were generated, and designated as 1D5 and 6A12. Isotyping revealed that the mAbs 1D5 and 6A12 were of the IgM class by determining with mouse monoclonal antibody isotyping reagents kit. These two mAbs could specifically react with IBV but not with NDV, AIV H5 or H9. The cross-react analysis detected that the mAb 1D5 had low cross-react with CK/CH/SCYA/10I and CK/CH/SCMY/10I, and the cross-react was lower than mAb 6A12.The eighth generation allantoic fluids containing IBV-Sczy3 strain was used to infect CEK cells to establishment the Sczy3 CEK cell adapt line, and the Sczy3 CEK cell adapt line was named as Sczy3-C. The 20th generation of the Sczy3-C was used to determinate the TCID50, the result was 10-5.64/0.2 mL. End-point neutralizing assay of the mAbs 1D5 and 6A12 indicated that the mAbs were the neutralizing monoclonal antibodies, and the 50% end-point neutralizing titers were 1:40.6 and 1:44.7, respectively.In order to identify the corresponding epitopes of mAb 1D5 and 6A12, the mAbs against IBV S1 protein were used as target moleculars to screen the 12-mer random peptide phage display library by biopanning. After 3 round, each round of increasing Tween-20 concentration and reducing the target molecule coating concentration to increase screening intensity, ten individual phage clones of each mAb were selected and amplicated and assayed for target binding using an sandwich ELISA. The positive phage clones DNA were amplified by PCR and analyzed the core sequences. Both of mAbs 1D5 and 6A12 were obtained 3 sequences. Comparison of these sequences with the sequence of S1 protein of Sczy3 showed that homology of 1D5 occurred at amino acids 87PPQGMAW93, and 6A12 occurred at amino acids:412IQTRTEP418. Homology analysis with 32 IBVs classified into 8 genotypes found that the positions in the motif 87PPQGMAW93 were highly variable, especially among the genotypes of CK/CH/LSC/99 I-type, proventriculus-type and the TW-type. and the positions in the motif 4I21QTRTEP418 were conserved. only be different with JP-Type. The oligopeptides containing the epitopes of S1 protain pET-sel (77aa-107aa) and pET-se2 (399aa-424aa) were prokaryotic expressed, and identified with mAbs 1D5 and 6A12 by Western blot analysis. The result showed that the recombinant proteins pET-sel and pET-se2 could interact with mAb 1D5 and mAb 6A12 respectively. All the results confirmed that the motifs 87PPQGMAW93 and 412IQTRTEP418 were two linear neutralizing B cell epitopes of the S1 protein of Sczy3.The present research enriched the neutralizing monoclonal antibody library and expounded the molecular antigenic structure of the S1 glycoprotein of IBV. It will be useful for understanding the relationship between the antigenic structure and function of the S1 glycoprotein, and the mechanisms of the genetic variation. It also consolidates the basis for establishing the epitope vaccine and epitope-basees diagnostic methods.