Expression Of NP Protein And Preparation Of Monoclonal Antibodies Against NP Protein Of H3N2Subtype Canine Influenza Virus

Canine influenza is caused by Canine Influenza Virus (CIV) H3N2subtype and is a newly identified canine disease. All kinds of dogs could be infected and show similar respiratory symptoms such as fever, cough, rhinorrhea, anorexia and depression, etc. After decades of variation and adaptation CIV is a spreading with a very rapid rate and is a treat to the global public health. Pet dogs carry CIV but show not obvious or inapparent signs, therefore playing an important role in our modern lives with the development of society. In addition, the period of virus shedding is very short and it is quite difficult to isolate CIV in pet dogs. In this study, the coding fragment of H3N2SIV NP gene was amplified by PCR, using a pair of primers with specific enzyme digestion sites. Then it was cloned into plasmid pMD18-Simple-T vector. After sequencing the correct plasmid was digested and cloned into the prokaryotic expression vector pET32a-(+).BALB/c mice were inoculated with0.3ml of the mixture of purified protein pET-NP with Freund’s adjuvant in a1:1ratio. After the fourth immunization, splenocytes from the immunized mice were fused with SP2/0myeloma cells (Strengthen the immune3days before the fusion). Positive hybridoma clones were screened by ELISA. After three cycles of subcloning, three monoclonal hybridoma cell lines against the NP of CIV H3subtype were generated. The MAb cell lines secrete antibody stability and have good specificity. The ELISA titer of the MAbs was from1:105to1:107and the MAbs belongs to IgG2b subclass, Kchain. After determination of antigenic activity of the purified ascites, it is showed that the antibodys is of high titers. The preparation of the MAbs against NP protein lays the foundation for the establishment of the CIV diagnosis. Furthermore, it serves as the basis for analyzing NP protein antigen epitopes.