Expression Of Canine Distemper Virus Nucleocapsid Protein Gene In E.coli And Establishment Of An Indirect ELISA For Detecting Viral Antibodies

The cDNAs of N genes of the CDV/R/20-8 strain were amplified by RT-PCR,cloned and sequenced. The recombinant plasmid pGEX-4T-1-N was transformed into E.coli Rosetta and the expression product-recombinant nucleocapsid protein of CDV was obtained under the optimized condition of host cell cultivation and IPTG induction, then the indirect ELISA was developed for detection of antibodies to canine distemper virus.Cloning and sequencing of CDV/R/20-8 strain N gene. The full-length of N gene encoding the Nucleocapsid protein of CDV/R/20-8 strain was amplified by RT-PCR with a pair of primers (p1/ p2) and cloned into plasmid pMD18-T. Molecular and phylogenetic analyses of N gene revealed that the CDV/R/20-8 strain genotype belongs to the lineage of vaccinal strain. The N gene is the most conservative region of CDV genome. What indicates that expressed N protein has similarity with natural Nucleocapsid protein on immunogenicity, which can be used as antigen for diagnosis. It laid a foundation for the development of genetically engineering vaccine, monoclonal antibodies and colloidal gold reagent paper of CDV. Prokaryotic Expression of N Gene of CDV/R/20-8 strain and primary application. The cDNA encoding N antigen was subcloned into pGEX-4T-1 and transferred into E.coil Rosetta. an 55 Ku expressed fusion protein was induced under 1.0mmol/L IPTG and 37oC, which was identified by SDS-PAGE . The CDV N protein could be expressed by pGEX-4T-1-N in Roestta at high level, up to 22.52% of the total protein of the induced bacteria. The recombinant protein was confirmed by Western-blot that there was a positive reaction with standard positive blood serum.Development of indirect ELISA to detect antibody of canine distemper virus. The recombinant CDV N protein was used as antigen. The purified N protein was used to be coated on the well of 96 well plate, each following step was optimized. As a result, an indirect ELISA was constructed to detect antibody against CDV. The various factors and conditions of ELISA were explored, and the optimal reaction conditions of ELISA were determined. It was shown that the optimal concentration of recombinant N protein for each well of an ELISA plate was coated with 1μg refined CDV N protein and the plate was locked by 10% rabbit serum . The optimal coating condition of recombinant N protein for ELISA was 4℃overnight, the dilution of serum sample was 1∶40.The blocking test for the reaction between the CDV positive serum and the recombinant N protein was carried out by using the CDV antigen. The results showed that CD virus could block the reaction completely. Meanwhile, it was confirmed that there was no cross reaction between the antibodies against other canine infectious diseases. The difference value among wells in a plate and among plates for ELISA was bothless than 5% , which showed the assay had a good retrievality. The results revealed that the indirect ELISA by the purified recombinant nucleocapsid protein has good specificity and sensitivity as well as high safety for the detection of CDV antibody in serum, it creates conditions for the development of monoclonal antibodies and colloidal gold reagent paper of CDV,which for clinical detected agents developing.