Preparation And Preliminary Application Of Anti-Rabies Virus Phosphoprotein ScFv-Alkaline Phosphatase Fusion Protein

Rabies is a natural foci of zoonotic diseases caused by rabies virus.Once infection,the mortality is as high as 100%.The rabies virus belongs to the Rhabdoviridae Rabies virus with a single strand of negative RNA,which enconds five proteins including glycoprotein(G),nucleoprotein(N),matrix protein(M),phosphoprotein(P)and RNA-dependent RNA polymerase(L).Since the developement of Rabies vaccine by Pasteur more than a century ago,the preparation of rabies vaccine and clinical using is very refined.However,the effective treatment for rabies Rabies disease is still lack.The detection technique of RV is necessary to monitor the natural infection and evaluate immune effect of vaccine.Phosphoprotein of Rabies virus is a highly hydrophilic protein acounting for 6%of the total viral protein.As a cofactor of viral RNA polymerase,Phosphoprotein is a multiple functional protein which plays an important role in the immune response by interacting with STAT1 on the interferon signaling pathway.Therefore,the preparation of single chain antibody fragment(scFv)against phosphoprotein is eeesntial to clinical diagnosis and treatment for Rabies.In order to express and purify the recombinant protein of rabies virus,first use the primer 5 software,the specific primers with EcoRI and SalI cleavage sites at both ends of the primers were designed by using the rabies virus phosphoprotein gene sequence retrieved in NCBI.The amplified gene was inserted into the PET-32a expression vector,and the positive clones were obtained by PCR and sequencing.Through the induction of gene expression and SDS-PAGE detection,the results showed that the recombinant protein was expressed in the inclusion body,and with protein purified,renaturation,dialysis and vacuum freeze-drying to obtain high concentration of rabies virus recombinant phosphoprotein.The results of ELISA and Western blot showed that the recombinant protein had 53 KD molecular weight and had good antigenicity.Furthermore,the mice were immunized with the obtained recombinant phosphoproteins to select the murine spleen cells,and the mouse myeloma cells with good growth were selected for cell fusion.The hybridoma cells were screened by indirect ELISA and indirect immunofluorescence,after antibody specificity and antibody concentration in the supernatant were also measured.Eventually we obtained a hybridoma cell line 1A4 which secreting anti-rabies virus phosphoprotein antibody.The positive hybridoma cells can prepared ascites.After purified and specific test the antibody secreted by the cell strain could effectively recognize the rabies virus protein.Finally,amplifing the heavy and light chain variable region genes of the antibody from the positive hybridoma cell lines,construction of Rabies Virus Phosphoprotein Single Chain Antibody Expression Vector.When designing a single chain antibody expression vector,we added the alkaline phosphatase gene at the carboxy terminus,the expression product is a fusion protein of single chain antibody and alkaline phosphatase.This fusion protein can be used as a direct antibody,detection without secondary antibody,save time,effort,easy to prepare,greatly reduce the cost of production.Western blot and indirect immunofluorescence assay showed that the anti-phosphoprotein scFv had a good antitussine protein binding effect.Through the preliminary detection of rabies clinical samples,found that the amount of single-chain antibodies,in competitive ELISA experiments have a good detection effect,the preparation of single-chain antibody has a better product development prospects.In this study,we successfully prepared rabies virus phosphoprotein,and on this basis,we successfully prepared a specific high monoclonal antibody and single chain antibody.Single chain antibody-alkaline phosphatase fusion protein as a direct antibody,not only for the experiment to save time and secondary antibody materials,but also in antibody detection applications,breaking the traditional antibody commercialization kit in the detection of species restrictions,we do not need to consider the difference between the corresponding secondary antibody problems,provide technical support for rabies virus diagnosis and evaluation of animal immune effects.