The Preparation Of Monoclonal Antibodies Against Csfv E0Protein And Establishment Of Immunocolloidal Gold Strip For CSFV

Classical swine fever(CSF), a highly-infectious disease, brings severe economic losses to the pig farming worldwide. It has been the most important infectious disease in our county many years. In order to control CSFV,many countries have paid a lot of manpower and money. In recent years, this disease showen many new characters.The incidence of this disease raised, the clinical representation became complex and the incidence of the immuned hogs raised. CSF is still the most important infection in our country.Based on the genome sequence of CSFV C stain, primers were designed to whole EO gene.714bp products were got by RT-PCR. The products were inserted to plasmid pET-28a for making recombinant plasmid and then transformed to host strain BL21. The positive clone identified by endonuclease digest were induced by IPTG. Target protein was Detected by SDS-PAGE. Both of recombinant are well expressed in the form of inclusion body with1.0mmol/L IPTG at37℃. An Ni+2affinity column is used to purify the inclusion body protein.Two hybridomas secreting antibodies against CSFV EO ptotein were established by fusing SP2/0with spleen cells from BALB/c mice immunized with purified EO ptotein, naming E01and E02. The Ig isotypes of mAbs were IgG2bκ. The ELISA antibody titers of the ascites were1:12800,1:25600respectively. The two mAbs did not react with two other swine virus in the specific analysis by indirect ELISA. This experiments laid the foundation for neutralizing monoclonal antibody therapeutics of CSFV and rapid detection kit.The purified monoclonal antibody E01was labeled with colloidal gold as capture antibody and the purified monoclonal antibody E02was immobilized on the test line as detection antibody, while a sheep anti-mouse IgG antibody was coated on the control line of the nitrocellulose membrane. Then the colloidal gold immunochromatographic strip for the detection of CSFV was assembled in regular sequence. Negative control were tested successfully. Detection roughly purified CSFV with this immunochromatographic test strip, control line is obviously red, while the color of test line is light.