Study On The Construction And Rescue Of The Rabies Virus SRV₉ Ψ Aera Delection Strain

Rabies virus(RV)is the sole cause of all warm-blooded animals and humans infected with rabies virus.A large number of studies show the virus gene can be modified and transformed in vitro,and the recombinant rabies virus can be rescued for further research on new attenuated RV vaccines.The construction of full-length infectious cDNA clone is an important key for the rescue of recombinant rabies virus.At present,the construction of full length infectious cDNA is mostly performed by a step by step enzyme digestion and ligation,then transfected into the eukaryotic cells by liposome method with complicated procedures,low efficiency of rescue and limitation in the new RV attenuated vaccine development.The shot sequences of hamRz and HdvRz were synthetized into predesigned PCR primers in the study,then sequences of HamRz and HdvRz were added into the whole genome of the 3’ terminus and 5’ terminus,respectively,by PCR amplification.According to the Gibson Assembly connect method,the primers PCR were designed and amplificated.The PCR products were structural protein genes with a small overlap sequence.The recombinant plasmid pcDNA3.1-L was constructed by the Gibson Assembly connect method,which divided a 6545 bp large transcription protein(large protein gene,L)gene into three sessions and inserted them to pcDNA3.1+ vector by one step.A predesigned NheI restriction site before the L gene was designed and used for assembling the recombinant plasmid pcDNA-HamRz-NPMGL-HdvRz which was the full length infectious cDNA.Then,the recombinant plasmid pcDNA-HamRz-NPMGL-HdvRz were transfected in BHK-21 cells with other five auxiliary expression plasmids and the recombinant virus was rescued.The recombinant Rabies virus SRV9 deleted pseudo gene Ψ region was identified by RT-PCR,indirect immunofluorescence,Western-blotting and electron microscopy.The results were shown(1)the full length infectious cDNA of Rabies virus SRV9 deleted pseudo gene Ψ region was successfully constructed;(2)The recombinant plasmid pcDNA-HamRz-NPMGL-HdvRz had been transfected in BHK-21 cells with other five auxiliary expression plasmids and obtained Rabies virus SRV9 without pseudo gene Ψ region identified by RT-PCR;(3)The recombinant Rabies virus could be specifically bonded with rabies positive serum and had good antigenic characteristics identified by indirect immunofluorescence assay and Western-blotting test,respectively,and(4)The rescue virus articles had a typical bullet characteristics under the electron microscope.The rescue method of Rabies virus SRV9 deleted pseudo gene Ψ region is a good tool for further study on virulence attenuation of RV,construction of vector RV and development on new multiple attenuated rabies vaccines.