Cloning And Prokaryotic Expression Of N Protein Gene Of Canine Distemper Virus And Antigenicity Of The Recombinant Protein

Canine distemper is a kind of infectious and fatal disease which affected on the development of special strain cultivation industry, caused by canine distemper virus infecting canine and other carnivora animal. The clinical features are diphasic fever, roseola, conjunctivitis, leucocytopenia, and central nervous system damage. It is particularly important to prevent and early diagnose, because now it doesn't have specific medicine to cure. The following three studies were conducted in the present article.1 Cloning and sequence analysis of N protein gene of canine distemper virus One pair of the special primers were designed by oligo6.0 software according to the N protein gene sequences of canine distemper virus (CDV) published in GenBank. A 1 572bp CDV N protein gene fragment was amplified by RT-PCR from the total RNA template in the whole blood samples infected by CDV, and it was cloned into a pMD18-T vector to construct pMD18-CDV N recombination plasmid. Sequence analysis showed that the total length of CDV N protein gene was 1 572 nucleotides from start codon ATG to stop codon TAA, and the homology of the nucleotide sequence was 93.9%-99.1% compared with that published in GenBank.2 Prokaryotic expression of N protein gene of canine distemper virus and reactionogenicity study of recombinant proteinThe cloned N protein gene from CDV was subcloned into expression vector pGEX-4T-2 and the recombinant plasmid pGEX-4T-2-CDV N was transformed into E.coli BL21(DE3)for sequencing, identifying and expressing with IPTG. Western blot analysis showed that a recombinant protein of 84kDa was expressed which could be recognized by antiserum against CDV, and the recombinant protein (GST-CDV N) possessed strong reactionogenicity.3 Optimization of prokaryotic expression conditions of N protein gene of canine distemper virus, purification of recombinant protein and its immunogenicity studyIn order to improve the dissoluble expression level of GST-CDV N recombinant protein in E.coli, various expressing conditions of GST-CDV N recombinant protein in E.coli were studied. After considerable expression at the optimal expressing conditions, GST-CDV N recombinant protein was purified by GST-Sepharose 4B affinity column to obtain the highest level dissoluble recombinant protein. The recombinant protein (GST-CDV N) was proved to possess strong immunogenicity in immunized mice.CDV N protein gene was successfully cloned and expressed in this study. The purified recombinant protein has strong antigenicity. It is possible to establish the ELISA detection system, and this lay the foundation for developing a subunit vaccine of CDV.