Expression Of Receptor Binding Domain Protein Of MERS-CoV In Insect Cells And Its Immunprotective Analysis

Objective: To prepare the recombinant S1 and RBD protein of MERS-Co V in the baculovirus expression system and evaluate the immunogenicity of purified recombinant protein. To produce and identify monoclonal antibody neutralizing MERS-Co V with recombinant S1 protein as antigen.Methods:(1) The recombinant bacmid with S1 gene of MERS-Co V inserted was constructed, which was followed by transfection into Sf9 cells to package recombinant baculovirus. The Sf9 cells were infected by recombinant baculovirus seed derived from the third passage. Cell culture supernatant with the recombinant S1 protein was collected and purified by nickel column affinity chromatography. Following vaccination in BALB/c with purified recombinant S1 protein, the S1-specific Ig G antibody inmouse sera were measured by ELISA. The neutralizing assay was performed and analyzed by pseudo-MERS-Co V and MERS-Co V. The BALB/c mice were immunized with recombinant S1 protein of MERS-Co V, and the fusion of lymphocyte and myeloma SP2/0 cells was performed by conventional method. After the screening of positive clones by ELISA, the hybridoma cell lines with stable secretion of specific antibody were obtained through subclonal method. Ascites antibodies were produced and purified, followed by characterization identification. Pseudovirus- and live-virus-based neutralization assay were carried out to screen monoclonal antibodies with neutralizing activity against MERS-Co V.(2)The protective functional domain of 355-590 aa in S1 was predicted after the analysis of MERS-Co V S1 sequence, secondary structure and flexibility companied with the crystal structure analysis on MERS-Co V RBD、MERS-Co V and the binding of RBD to DPP4 receptor. After the construction of recombinant bacmid inserted with RBD、RBDFd gene of MERS-Co V, Sf9 cells was transfected with recombinant bacmid to package recombinant baculovirus. Then the Sf9 cells were infected with recombinant baculovirus seed derived from the third passage. Cell cultures supernatants with recombinant proteins were collected and purified by nickel column affinity chromatography. Following vaccination with purified recombinant protein, the RBD-specific Ig G antibody and RBDFd-specific Ig G antibody responses in BALB/c mouse sera were measured by ELISA, and the neutralizing antibodies were detected by pseudo-MERS-Co V neutralizing assay. Results:(1) The recombinant baculovirus isolate expressing recombinant S1 protein was obtained and the recombinant S1 protein was successfully expressed and purified in insect cells. Following three vaccinations with recombinant S1 protein, titer of S1-specific Ig G antibody in mouse sera reaches 1:102400, and inhibition percentage against pseudo-MERS-Co V can be still over 50% at 1:5120 dilutions.(2)Ten hybridoma cell lines with stable secretion of specific antibody were obtained. 6 of ascetic monoclonal antibodies belonged to the Ig G1 subtype, 2 of them belonged to the Ig G2 a, 2 of them belonged to the Ig M subtype. All the ascites have high antibody titers reaching to 10000. 1 strain of monoclonal antibody was further proved to have neutralizing activity.(3) The recombinant baculovirus isolates expressing recombinant RBD、RBDFd protein were obtained and the recombinant RBD、RBDFd protein were successfully expressed and purified in insect cells. Following three vaccinations with recombinant RBD protein, titer of RBD-specific Ig G antibody in mouse sera reaches 1:6400, and inhibition percentage against pseudo-MERS-Co V below 50% only at 1:3200 dilutions. But after booster vaccinations with recombinant RBDFd protein, titer of RBDFd-specific Ig G antibody in mouse sera reaches 1:102400, and inhibition percentage against pseudo-MERS-Co V can be over 50% only at 1:12800 dilutions.Conclusion: Recombinant MERS-Co V S1 protein expressed in insect cells has ability to induce good immunogenicity with high titers of neutralizing activity, and one strain of monoclonal antibody with good neutralizing activity against MERS-Co V was obtained. Recombinat MERS-Co V RBD protein expressed in insect cells has weak ability to induce good immunogenicity and neutralizing activity.However, recombinant MERS-Co V RBDFd protein expressed in insect cells has ability to induce strong immunogenicity with high titers of neutralizing activity. The study lays a foundation for the development of recombinant vaccine against MERS-Co V.