Establishment Of Reverse Genetics Operation System Of Peste Des Petits Ruminants Virus Clone9 Strain

Peste des petits ruminants(PPR)is a highly contact and fatal disease of small ruminants such as goats and sheep caused by peste des petits ruminants virus(PPRV).PPR is a notifiable disease listed by the world organization for animal health(OIE),and it is also a class of animal disease stipulated by China,which seriously endangers the development of animal husbandry.So far,immunization is the only effective measure to prevent and treat the disease.This study intends to establish the reverse genetic operation system of PPRV Clone9 strain,and explore the potential of PPRV Clone9 strain as vaccine vector by inserting foreign gene enhanced green fluorescent protein(EGFP).The sequence of the PPRV Clone9 strain preserved in our laboratory was determined,and the fulllength PPRV Clone9 was amplified in 6 segments.The full-length c DNA infectious clone plasmid of the viral genome was constructed by Hdv Rz,and the T7 RNA polymerase promoter,termination The nuclease and butyl ribozyme sequences were introduced into both ends of the PPRV Clone9 genome;At the same time,the helper plasmids expressing N,P and L were constructed,and the above plasmids were co transfected into BHK cells stably expressing T7 RNA polymerase through liposome mediated.The virus(r PPRV Clone9)was successfully rescued.The virus infected Vero cells could produce the same cytopathy as the parent virus,and the proliferation dynamics was consistent with the parent virus.EGFP gene was inserted into the noncoding region between P and M of r PPRV Clone9 genome.The full-length c DNA of the virus genome containing foreign gene EGFP was cloned into BHK-T7 cells expressing T7 RNA polymerase,and the green fluorescence was observed under purple excitation light.After identification by RT-PCR,indirect immunofluorescence and Western blotting,the rescued recombinant virus(r PPRV-EGFP)could be stably passaged on Vero cells,and the proliferation dynamic was similar to that of the parent virus,which proved that r PPRV Clone9 could be used as a vector to express foreign proteins.