Construction Of Recombinant Rabies Virus SRV₉ Strain Of Echinococcus Granulosus EgM123 Gene

Rabies is a highly lethal infectious disease caused by rabies virus infecting the central nervous system of humans and animals,and it is a worldwide public health prevention and control disease.Hydatid disease,also known as Echinococcus granulosus,results in the formation of Echinococcus granulosus cysts in multiple organs,causing a serious zoonotic chronic parasitic disease.During the transmission of rabies and hydatid disease,dogs are not only the main vector of rabies transmission,but also the main host of hydatid disease transmission.Immunization of canines is a key link in controlling the spread of these two diseases.The currently produced echinococcosis vaccines are mainly subunit vaccines constructed by using Eg95 recombinant protein as antigen to prevent and control Echinococcus granulosus infecting intermediate hosts（sheep）and have achieved satisfactory results.Vaccine development in dogs.In this study,in order to construct the recombinant rabies virus SRV₉strain vaccine strain of EgM123gene by reverse genetics technology,the whole genome sequence of rabies virus SRV₉strain was used to synthesize the rabies virus structural gene N,P,L,N+P+M fusion gene and rabies virus respectively.The viral G gene was inserted into the foreign gene EgM123 and eGFP fusion gene of Echinococcus granulosus,and the fusion gene N+P+M and G+EgM123+eGFP gene and the structural gene L gene were connected to pc DNA3.1（-）Infectious clone full-length c DNA of EgM123 gene recombinant rabies virus SRV₉strain was constructed in the expression vector.The results of plasmid double-enzyme digestion and sequence sequencing showed that the sizes of rabies virus N,P,L,N+P+M,G+EgM123+eGFP gene fragments were1 541,1 107,6 644,3 160,and 3 256,respectively.bp;the size of the constructed full-length c DNA is 12465 bp.The results of sequencing and alignment of each gene sequence were 100%.In this study,the helper plasmids N,P and G gene inserted into the recombinant plasmid of exogenous gene EgM123 and green fluorescent protein eGFP gene identified by sequencing,In order to detect the effect of protein expression in BSR cells and the identification of protein immunogenicity,The successfully constructed pc DNA3.1-G+EgM123+eGFP and helper plasmids pc DNA3.1-N,pc DNA3.1-P Transfection of BSR cells by lipofection,make it expressed in BSR cells,and observed by fluorescence inverted microscope,protein electrophoresis,Western blotting Identification of Fluorescent Protein Expression of Fusion Proteins,Fusion protein molecular weight,Immunogenicity and protein expression of N and P proteins of rabies virus SRV₉strain.pc DNA3.1-G+EgM123+eGFP recombinant plasmid transfection results show,at 48 hours after transfection,the expression of green fluorescent protein was observed with a fluorescent inverted microscope.Displayed by protein electrophoresis and WB results,The recombinant plasmid is expressed in BSR cells,Transfer protein gel to PVDF membrane,Rabies virus G protein monoclonal antibody,EgM123 polyclonal antibody,GFP monoclonal antibodies were incubated separately,Antigen-antibody binding bands are visible at 120 KDa.The transfected helper plasmids pc DNA3.1-N and pc DNA3.1-P were identified by WB,Structural proteins N and P are at 55 KDa and 40 KDa,Antigen-antibo-dy binding bands can be seen on the left and right.In this experiment,the rabies virus SRV₉strain was used as the vector,The constructed SRV₉-EgM123-eGFP full-length c DNA recombinant plasmid and the helper plasmids pc DNA3.1-N,P,L were co-transfected into BSR cells expressing RNA polymerase,The recombinant virus"SRV9-EgM123-eGFP"was rescued.The rescued recombinant virus was detected by fluorescence inverted microscope,RT-PCR,WB assay,and transmission electron microscope,Visible fluorescent foci are visible under a fluorescent inverted microscope,RT-PCR and WB results showed that structural genes P,G and foreign gene EgM123 could be detected in the rescued recombinant virus,The rescued strains were found to have typical rhabdovirus particle morphology under transmission electron microscope.The rescued recombinant virus of EgM123 gene of Echinococcus granulosus with the G gene of rabies virus carrying enhanced green fluorescent protein（eGFP）will be rescued,and the vaccine strain will be provided for the development of"rabies and hydatid disease dual gene recombinant vaccine",for more rapid and accurate detection of vaccine titers.