Cloning And Expression Of N Gene Of CDV And Elementary Establishment Of Indirect ELISA To Detect The Antibody Of CDV In Blood Serum

Canine distemper (CD) is an acute, highly contagious disease, caused by canine distemper virus(CDV). It causes serious loss for the industroy of canine and other peltry animals in the world. Canine distemper virus (CDV), whose genome encodes six structural proteins and non-structural protein(C-protein). Nucleocapisid protein is an important structural protein, which is coded by Nucleocapisid gene. It has T cellular epitope. Nucleocapisid protein have important function in the process of generation including assembly, replication, transcription. It also has considerable contribution to cellular immunity. So the significance is enormous to study and research Nucleocapisid protein of CDV.In order to explore the possibility of using nucleocapsid protein of CDV virus as a kind of diagnostic antigen, nucleocapsid protein gene was amplified middle conserved region gene of nucleocapsid protein from recombinant plasmid pcDNA-N by PCR, It was incised with EcoRI and SalI, inserted into prokaryotic expression vector pGEX-6P-1 , and demonstrated by testifying sequence. The result showed that it was fundamentally accord with that of nucleocapsid protein of Onderstepoort strain that has been reported. There recombinant plasmid pGEX-6P-1-N that had designed was introduced into competent cells of BL21(DE3) and then induced for five hours to express by IPTG. The expression product was assayed with SDS-PAGE,Western-blotting and IFA,There recombinant protein account by to 14.6% of total amount of bacterial protein existed mainly in the superanant fluid after boiling, and this recombinant protein was verified to react positively with rat anti-dog polyclonal anti-serum.The method of indirect ELISA to detect CDV antibody in blood serum was established, using recombinant nucleocapsid protein as envelope antigen. Tests were carried out to optimize the process, containing the most compatible density, the best dilution of blood serum, the most suitable working concentration of anti-antibody marked by HRP and so on. It showed that the antigen optimal density is 0.525μg/100μL, the best dilution of blood serum is 1:40 and the most suitable working concentration of anti-antibody marked by HRP is 1:2000.Indirect ELISA was tested by the experiments of reproducibility, specificity and so on. The results show that degree of variation is low; diagnostic antigen has no cross reaction with positive blood-serum of Canine parvovirus or Canine parainfluenza virus.In conclusion,The product of expression of recombinant nucleocapsid protein as envelope antigen was used in the establishment of indirect ELISA, which has considerable contribution to diagnosis and serosurvey of CDV.