Development Of The Monoclonal Antibody With Specificity To Phosphate Protein Of Newcastle Disease Virus By Gene Immunization

In order to produce the monoclonal antibody against phosphate(P) protein of Newcastle disease virus(NDV), mice were immunized with the recombinant plasmid pcDNA3.1(-)-P, and the fusion protein(GST-P) expressed from the recombinant plasmid pGEX-6p-l-P was used as screening antigen to screen hybridoma cells. The P gene of NDV strain ZJ-1 was amplified by polymerase chain reaction (PCR) from the plasmid of TNDV4 containing the full-length sequence of NDV ZJ-1, and then was cloned into the expression plasmid pcDNA3.1(-). PCR and restriction endonuclease analysis were used to identify the recombinant plasmid, and the results indicated that the fragment of interest was correctly inserted. Then the recombinant was transfected into the COS-1 cell. The indirect immunofluorescence assay(IFA) was used to identify the expression of the P gene 72 hours post-transfection, and the result showed that the P protein was expressed. The 6-week-old female BALB/C mouse was thereafter immunized with the recombinant 100μg . Spleen cells collected from immunized mouse were fused with SP2/0-Ag-14 myeloma cells 7 days after four immunizations.The recombinant plasmid pGEX-6p-l-P was constructed. PCR, restriction endonuclease analysis also were used to identify the recombinant plasmid containing GST-P fusion gene. Then the plasmid was transformed into E. coli BL21. Soluble GST-P fusion protein was expressed in BL21 after induced by IPTG at 37℃, which was confirmed by SDS-PAGE and Western-blot. The indirect enzyme linked immunosorbence assay(ELISA) was established with the fusion protein as screening antigen to screen hybridoma cells and the method of limited dilution was performed to subclone the positive clones. One positive clone was obtained, designed as 2G8. The immunoglobulin subtype of the McAb 2G8 was identified as IgM. The specificity of theMcAb 2G8 was finally characterized by IFA and Western-blot.