The Establishment Of Novel Live Virus Independent Neutralizing Antibody Detection Method Based On The Interaction Between MERS-CoV RBD And DPP4 Receptor

The Middle East respiratory syndrome(MERS)is a novel highly pathogenic zoonosis caused by Middle East respiratory syndrome coronavirus(MERS-CoV)which break after the severe acute respiratory syndrome(SARS)in 2002.It originated from bats and caused human severe pneumonia,shock and organ failure after infection,leading to more than 35 percent fatality rate.There are no approved drugs and vaccines in the treatment and prevention of MERS clinical infection,thus establishing fast MERS-CoV neutralization antibody detection and classification method,as well as the further development of MERS therapeutic antibodies and vaccines has become the key to prevent and control of MERS.Neutralization antibody detection is the most commonly method in disease diagnosis,therapy and prevention.Because the MERS is a highly pathogenic zoonosis,its pathogenic operations must be in P3 biosafety laboratories or above,the traditional MERS-CoV neutralizing antibody detection method relying on live virus greatly limits the neutralization test applications in the study of MERS.The receptor binding domain(RBD)of MERS-CoV Spike protein(S)can specifically recognize and bind to the receptor dipeptidyl peptidase(DPP4)on cell membrane,and mediate viral cell entry and infection.In addition,RBD is neutralizing epitopes rich domain,and the neutralizing antibody targeting to RBD could specifically block MERS-CoV cell infection and RBD binding to DPP4 receptor expressing cells.Based on the forementioned principle,this research established two novel live-virus independent MERS-CoV neutralizing antibody detection methods with flow cytometry,by replacing live MERS-CoV with human IgG1 Fc and MERS-CoV RBD fusion protein(rRBD-Fc),and taking DPP4 expressing cell line Huh-7 or DPP4 conjugated magnetic beads as loading vector.1.This study used the commercial eukaryotic expression vector pFUSE-hIgG1-Fc which has Fc labels and multiple cloning site of EcoR I and Bgl II,through synthesizing RBD sequence with EcoR I and Bgl II at terminals,constructing the recombinant expression vector pFUSE-RBD-hIgG1-Fc.The recombinant expression vector has been transfected into 293T cells and the recombinant protein rRBD-Fc was purified with protein A resin.The recombinant protein was further identified by SDS-PAGE and Western blot.2.The Huh-7 has been incubated with the gradient dilution rRBD-Fc,and then coupled with FITC-labeled anti-Fc antibody.Flow cytometry detection results showed that rRBD-Fc has the capacity specifically binding to the MERS-CoV receptor DPP4 on the surface of Huh-7 cells,and the optimal binding dose is 0.25?g/1×10~5 cells.3.After incubation the rRBD-Fc with RBD-specific neutralizing antibodies,RBD-specific non-neutralizing antibodies,RBD immune serum,and MERS-CoV unrelated antibodies,the mixtures were then incubated with Huh-7 cells to detect whether the known serum or antibodies could block rRBD-Fc binding to Huh-7 cells by flow cytometry.The result showed that the novel live-virus independent neutralizating antibody detection method could test the neutralization activity of the above four types of antibodies or serum qualitatively and quantitativelyparallel test rRBD-Fc blocking acitivity with Huh-7 cells.4.In order to identify whether the novel MERS-CoV indenpent neutralizating antibody detection method was in consistent with the traditional neutralizating antibody detection method,the novel method was applied to detecting 12 strains of nanobodies,6 samples of rRBD-Fc immune mice sera,6 samples of normal mice sera,10 samples of camal serum and10 samples of health human serum.The parallel test results suggested that the novel live-virus independent MERS-CoV neutralizing antibody detection methods were in consistent with tradiational MERS-CoV tradional neutralization test.5.Considering the flow cytometry test results could be interfered by the state of Huh-7cells,the DPP4 protein has been coupled in the surface of magnetic beads to replace Huh-7cells.The result showed that the optimal dose of rRBD-Fc binding to DPP4 coupled magnetic beads(0.25?g DPP4/2?l magnetic beads)was 0.4?g.By incubation rRBD-Fc with MERS-CoV RBD specific neutralizing antibodies(e.g.,N10,hMS-1,RBD first-immune mice serum),RBD specific non-neutralizing antibodies(N7)and MERS-CoV unrelated serum(such as normal serum and mouse immune serum)respectively,the result showed that the binding between rRBD-Fc and DPP4 coupled magnetic beads could be blocked by MERS-CoV RBD specific neutralizing antibodies,which suggested the tradional neutralization test could be replaced by the MERS-CoV live-virus independent neutralizing antibody detection method.In order to overcome the limitation that the traditional neutralizing antibody detection method relying on live virus,this study established two new neutralizing antibody detection methods of MERS-CoV without virus and/or cells.Compared with traditional neutralization antibodies detection method,the new methods established in this study are safe,easy to operation and time-saving,which are expected to be widely used in the diagnosis,treatment and prevention of MERS in the future.