Cloning And Expression Of Canine Distemper Virus Nucleocapsid Protein Gene

Canine distemper is a highly infectious,frequently lethal disease in dogs and other camivore.In order to develop DNA vaccines which are safe,convenient for use for canine distemper,we designed a series of experiments to reach the goal.The templates were produced from the reverse transcription reaction that RNA was isolated from OP-CDV infected Vero cell culture.Two pairs of primers had been designed and synthesized based on the Nucleocapsid(N) protein gene of the onderstepoort strain of canine distemper virus(CDV). 822bp and 897bp fragments of N gene were amplified by polymerase chain reaction(PCR) respectively. PCR products were cloned into the expressing plasmid vector pGEX-6P-l. The recombinant was verified by restriction endonuclease analysis and sequenceing. Then two recombinants were transformed into the host strain BLai and expressed by 1.0mM IPTG induced at 37#. The expression products were identified by SDS-PAGE and found to be 52kDa and 54kDa band in size as two fusion proteins with glutathione S transferase (GST) protein. The specific bands of expression were excised from the gel mixed and injected into the mice once a week and last for five weeks. The specificities of antibody were detected positivity against CDV-infected Vero cell by indirect immunoinflurescant assay(IFA). The results of the study showed that in vitro expression of N gene maintained some antigenicity of CDV.Designed a pair of primers which contain two restriction sites,î–©oR I and BamH I, and canine distemper virus N gene were amplified from the OP-CDV infected Vero cell culture by reverse transcription polymerase chain reaction(RT-PCR).The purified PCR products and empty plasmid pcDNA3.1(-) were digested by EcoR I and BamH I ,ligated,and transformed competence E.coli DHs ?.Positive recombinants were indentified by Ampicillin and restriction endonuclease digestion. pcDNAa i- N recombinant has been constructed successfully.The recombinant plasmid was transfected into COS-7 cell with lipofectin. Immunoinflurescant assay showed the recombinant was transient expressed into the COS-7 cell.Mices were inoculated with DNA and PEI by intramuscular injection,which produce the high antibody of ELISA.The recombinant plasmids will be useful for the generation of DNA vaccines.