Cloning And Sequence Analysis Of H Gene,F Gene And N Gene Of Canine Distemper Virus Isolated In A Fox In Tai'an And Establish A TaqMan-based Real-time RT-PCR Method For Detection Of Canine Distemper Virus

Three pairs of primers were designed based on the H protein gene,F protein gene and N protein gene of the strain of canine distemper virus according to Genbank. And using RNA extracted from the Taian isolated(CDV-FOX-TA)as template, the H protein gene ,F protein gene and N protein gene were amplified with RT-PCR. The PCR products were cloned into PMD18-T vector, and the positive recombinants of the gene obtained were then sequenced and analyzed. And at the same time inorder to establish a TaqMan-based real-time RT-PCR method for detection of canine distemper virus ,one pair of primers and a TaqMan-based probe were designed and synthesized based on the M protein gene of canine distemper virus according to Genbank. The primers, probe and the reactive condition were optimized to improve the sensitivity and specificity of the method.The result showed that:The H gene of CDV-FOX-TA contained one 1824bp large open reading frame, and the virus had the lowest identity with the vaccine strains (CDV-Onderstpoort strain and CDV-Convac strain),only 89.2% and 90.6%, respectively. However, it had 98.6% identity with the stranded strong strain CDV-A75/17,and had 98.7% identity with Chinese strain CDV LP,which was isolated from lesser panda.The ORF in the F gene of the CDV-FOX-TA strain was 1889bp,the major difference among various CDV strains was the long signal peptide domain while the immature protein F0 exhibited high identity, and the RRQRR ,all of the 13 serine residues,4 protential asparagines glycosylation sites and the 2 hydrophobic regions supposed to affect the fusion function were high conservative. Phylogenetic analysis showed that the strain CDV-FOX-TA, CDV-A75/17 and the strain CDV-5804P have the nearest inherit distance. Based on the information we inferred that the strain CDV-FOX-TA, CDV-A75/17 and the strain CDV-5804P were all strong strains, perhaps they have the same ancestor.The large open reading frame length of the N gene of CDV-FOX-TA was 1572bp and encoded 523 amino acids. It had 96.0% and 95.9% identity with the vaccine strains CDV-Ondertepoort and CDV-Convac, respectively. However, it had high identity with wild strains, between 98.4% and 98.9%.The major diffence among CDV-FOX-TA and infirmness strains were N terminal and C terminal, while the region between N terminal and C terminal was showed to be high conservative. Phylogenetic analysis showed that N gene was more conservative than H gene and F gene. Based on the information we inferred that used N gene as aimed gene of nucleic acid vaccine and recombine vaccine in immuno-prevention of canine distemper perhaps had more availability.The specificity of the method was high and the result of reproducibility test was good and the sensitivity of the method was 0.01TCID50.38 samples had been tested by real-time RT-PCR. The results were compared with electron microscopy, RT-PCR and they were greatly coincident. It showed that this method may be used in clinical diagnosis, epizootic study and laboratory research.