| African swine fever (ASF) is a highly contagious and fatal hemorrhagic viral disease of domestic pigs, and caused by African swine fever virus (ASFV). Since the mid-twentieth century, ASF has been disseminated in dozens of countries in Africa, Europe and America, and in recent years it spread to Georgia, Armenia, Azerbaijan and Russia. Once ASF invaded to our country, it would cause great loss.VP73protein is abundant in the virus-infected cells and the most conservative antigenic protein among ASFV, which is encoded by the B646L gene, and with the molecular weight about73ku. VP73is an important component of the icosahedral capsid of the virus particles, and produced in the late stage of viral infection, and accounting for32%of the protein of whole virus particles. VP73protein in African swine fever virus plays a very important role in the process of binding and entering the receptor cells, and has a vital role for formation of virion capsid. Most strains of African swine fever virus virulence are very strong, but with the very low immunogenicity, only a few proteins are immunogenic. Studies have shown that the corresponding epitopes are quite conservative among VP73antibodies which are induced by the different regions of the ASFV strain. The antigenicity of the VP73protein is very stable, and is often used as a serological test and immune agents.Based on the known antigenic region of VP73gene sequence in GenBank, we designed a pair of primers, and amplified a VP73gene fragment with612bp by Polymerase Chain Reaction (PCR) from the Positive plasmid pMD18T-vp73, and cloned into plasmid pET32a(+). The recombinant plasmid was named as pET32a-vp73. The expression vector of pET32a-vp73was transformed into E.coli strain BL21(DE3). After inducing by IPTG, the recombinant VP73Protein was expressed and showed with expected molecular weight43ku in12%SDS-PAGE. The Western-blot result showed that the recombinant VP73Protein had the antigenic characteristic of ASFV. We optimized the expression condition of VP73protein. The optimized conditions were:0.8mM IPTG, incubating5h at37℃. After the centrifugation of the induced E.coli strain BL21(DE3) which contained expression vector of pET32a-vp73, Analyzing the supernatant and sedimentation with12%SDS-PAGE. The result showed that the recombinant protein was expressed in the inclusion body. The recombinant VP73protein was purified with the Ni-NTA affinity chromatography kit with the steps, including cell breakage, inclusion body washing, inclusion body solubilization with6M urea solution, and on column purification.The monoclonal antibody (MAb) against Protein VP73of ASFV were produced by fusing mouse myeloma cells (SP2/0) with BALB/c mouse spleen cells which is immunized with the prokaryotic expressed and purified protein VP73of ASFV. The optimal working concentration of the indirect ELISA for detecting and screening positive hybridoma cells was established by continual tests. Polystyrene microplates were coated with the purified VP73protein at the concentration of2μg/mL. The positive and negative serum was diluted1:1000. All the incubation steps above were done at37℃for60min. Indirect ELISA was used to screen hybridoma cells and limited dilution method was performed to subclone positive clones. We got one positive hybridoma cell line, and named as1C8after3sub-cloning. The hybridoma cell strain can steadily secrete antibodies after culturing consecutively three months and revitalization after two months cryopreservation. The antibodies are all IgG1subtype. ELISA titer detecting test showed that the titer of the hybridoma cell strain can reach102400. The MAb was against the VP73protein rather than the His-tag protein. The antibodies have not shown any cross-reactivity with other virus by the ELISA method. Competitive ELISA test showed that the MAb could compete with the ASFV infected serum product of pig. The preparation of the McAb will provide strong technical support for the establishment of etiological and serological detection methods of ASFV. |
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