Prokaryotic Expression and Monoclonal Antibody Preparation of Rabies Virus Phosphoprotein

Abstract

Background: Rabies is a zoonotic infectious disease that infects the human and animal central nervous system. Worldwide, especially in low-income countries, this disease is still a burden for public health. Among the rabies virus proteins, phosphoprotein plays a very important role in viral infection, and this research found that immunization of rabies virus vaccines could widely induce antibody responses against phosphoprotein, therefore rabies virus phosphoprotein may be a useful target for development of rapid and low cost serological diagnosis test, and therapeutic drugs. Objectives: The aim of this study was to prepare anti-rabies virus phosphoprotein monoclonal antibody, which is used for rapid detection and diagnosis of rabies virus infection. Methods: The phosphoprotein gene was amplified by the polymerase chain reaction (PCR), and sub-cloned in a his-tagged prokaryotic expression vector to generate a recombinant plasmid named pET-32a-RABV-P. Recombinant RABV-P was induced by isopropyl-β-D-Thiogalactopyranoside (IPTG), and then purified by the Ni-NTA purification system. Immunization of BALB/c mice with the recombinant protein was performed, and the spleen cells of the immunized mice and SP2/0 myeloma cells were fused together to obtain the monoclonal cell strains, and then identification of the characteristics of the antibody by the enzyme linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay was done. Results: The prokaryotic expression vector of pET-32a-RABV-P was successfully constructed. The fusion protein was expressed in Escherichia coli Rosetta and purified. After cell fusion, a hybridoma cell line 1A4 was successfully obtained. The antibody titer of the anti-RABV-P ascites reached 256,000. The results of western blotting and indirect immunofluorescence assay indicated that 1A4 hybridoma cell line was able to produce specific antibodies against rabies virus phosphoprotein. Conclusions: Recombinant rabies virus phosphoprotein could be successfully expressed in E. coli. A specific monoclonal antibody against rabies virus phosphoprotein has been successfully prepared.