Cloning And Expression Of Rabies Virus G Protein And Preparation And Identification Of Monoclonal Antibody Specific To V Region Of G Protein

Rabies is a lethal zoonotic infectious disease,caused by rabies virus(RV)infection of the central nervous system.RV genome is a single negative strand RNA,which has five structural proteins,namely N protein?P protein?M protein?G protein and L protein.G protein plays an important role in viral structural proteins,which exists on the surface of the virusparticles:it is a major protective antigen which stimulate the body to produce the humoral immunity and cellular immunity;it plays an important role in the viral pathogenicity and tropism in the nerve,directly related to the virulence of virus.In this study,according to the complete sequence of G gene of the attenuated LBNSE strain of RV,the prokaryotic expression vector p ET-30a-G was constructed successfully,which can express RV G protein.Recombinant expression vector p ET-30a-G was transformed into Escherichia coli expression strain BL 21(DE3)and induced by IPTG.The SDS-PAGE and Western blot method is used to analysis its expression.The results showed that there was a large number of protein near the protein molecular weight of 67 KD.It added that G protein was correctly expressed by p ET-30a-G recombinant vector and has the corresponding protein reactivity.The results can be used to provide the basis for further research.Simultaneously,the further study is the eukaryotic expression of G protein of attenuated LBNSE strain and wild-type IMDRV-13 strain of RV.The eukaryotic expression vectors,be it p CAGGS-G and p CAGGS-13 G,were constructed successfully.The expression of recombinant vectors was identified by immunofluorescence and RT-PCR.The results showed that there was a specific fluorescent signal in immunofluorescence,and specific bands in RT-PCR,which indicated that the eukaryotic recombinant expression vectors had the ability to express G protein correctly.This study can be used for the identification of Mc Ab recognition sites in subsequent research.It can also provide research foundation for the further study of the biological functions of RV G protein.In order to prepare monoclonal antibody(Mc Ab)against the glycoprotein(G protein)of rabies virus(RV)and identify its binding ability,in this study,we used the polypeptide "PPDQLVNLHDFRSDEIEHLVVEE" conjugated to KLH to immune mice.A hybridoma strain designated 4D3 was successfully prepared,which belonged to Ig G2 b subclass with a ? light chain.The eukaryotic expression vector p CAGGS-G/p CAGGS-13 G were constructed and G protein was transiently expressed in vitro.The Mc Ab secreted by 4D3,as analyzed by Western blot and immunofluorescence assay,specifically reacted with the eukaryotically expressed G protein of the attenuated RV strain LBNSE,but not with that of virulent strain IMDRV-13.The corresponding sequence of immune peptide,LBNSE strain and IMDRV-13 strain were compared with each other,the 264 AA of G protein directly affects the binding of Mc Ab.It can be inferred that the Mc Ab can recognize the linear V region epitope of G protein.All these fundamental studies are important for the control and elimination of the rabies virus,to provide research basis for further revealing the biological function of rabies virus glycoprotein.The Mc Ab prepared in this study thus constitute a useful tool for identification of field isolates of rabies virus and structural and functional investigation of the G protein.Our laboratory has also designed a combination of other peptide sequences in order to obtain a full coverage of the antibodies combination.