Identification And Infectious Clone Of Canine Distemper Virus SH Strain

Background:Canine distemper disease is caused by canine distemper virus(CDV),it’s a acute and deadly infectious disease that made domestic dogs and many carnivores dead.The death rate can go to 80% and it is a serious harm to kennel and fur animal breeding industry.So far,not many virulent CDV strains have been established in our country,while it’s a major technical obstacle for invasion,spread study.Therefor,it is necessary to developinfectious clone of CDV.Method:(1)Grinding the tissue of infectious dog according to proportion 1:10,then infecting the BHK-SLAM culture that express SLAM after filtration.Collecting the virus and passaging cells till third generation at 48-60 h after infected, total RNA was exacted by RT-PCR after the collection of infectious cells to detect the virus. Draw viral growth curve and determine virus titer.Then massively proliferate virus and differential centrifugate to purify the virus.Using the electron microscope to identify the virus.(2)Amplifying the complete genomic sequences and H gene of CDV by RT-PCR technique,then stitching the sequence using bioinformatics.Complete genomic sequences was named CDV-SH and the H gene was named CDV-SH;(3)The CDV-N gene was amplified by PCR and cloned into expression vector pET-32a(+),and the recombinant plasmid pET-32a-N was transformed into E.coli BL21(DE3) cells before being induced by IPTG. CDV-N protein was purified by inclusion body. PCR amplified 1572 bp of the CDVN gene, the recombinant protein vaccinated into rabbit to produce polyclonal antibodies. The anti-serum was collected and detected by western blot specificity.(4)Eight specific primers were designed according to the complete genome sequence to develop reverse genetics systems of CDV.The system was based on vector pSK and was named pSK-CDV-SH.At the same time,we expressed N、P、L protein of CDV-SH as eukaryoyic expression plasmid named pCI-CDV-N、pCI-CDV-P、pCI-CDV-L.Then transfer the four plasmids into BHK-SLAM through the calcium phosphate transfection method. Collecting the virus and passaging cells till third generation at 72 h after infected,identifying the virus using IFA method,the result showed that we successfullydeveloped infectious clone of CDV-SH.Result:(1)We isolated the canine distemper virus and determine virus titer,the time of its peak is 48 h after infection and the peak titer is 10-4.8(2)We cloned and spliced the complete genome sequence of CDV-SH,the result of sequence analysis showed CDV-SH belongs to Asia-I;(3)We expressed the N protein and prepared polyclonal antibody with good specificity;(4)We developed infectious clone of CDV-SH.Conclusion:The isolation of CDV-SH and preparation of polyclonal antibody and development infectious clone of CDV-SH provide a plat to study the character of CDV.