The Study Of Interaction Of Vesicular Stomatitis Virus Glycoprotein With LGALS1

Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strandRNA virus, which belongs to Vesiculovirus Rhabdoviridae. Cows, pigs, horses andother mammals and their insect vectors are VSV’s natrual host. The clinical signs ofVesicular Stomatitis are water vacuoles and ulcers on the tongue, lips, cheeks,papillae, and coronet, which is similar to FMD, VE, SVD, therefore, it is easilymisdiagnosed in veterinary clinical diagnosis. VSV also infects human and causesflu-like symptoms. Thus, it is a disease of great significance in Public Health. TheVSV G protein is the main viral determinant of the entry, it enables infection of most,if not all, human cell types, and of organism as distant as zebrafish and Drosophila.The VSV G protein directly affects the adsorption process of a host cell, and virusbudding by combining the host cell surface receptors. Despite some literature of thestudy around VSV receptors, no effective G protein receptor has been reported. Tofind proteins that can interact with VSV G protein, therefore, will lay the foundationfor VSV receptor study, and has important significance in infection process andpathogenic mechanism of VSV.In former study, VSV-G has been used as bait to screen its interacting proteinby yeast two hybrid method, and pull-down was used to verify the interactionprimitively. On this basis, VSV-G gene was inserted into pEF1a-IRES-DsRed-Express2eukaryotic expression vector, to constructed recombinant pEF1α-VSV-G;The result of double enzyme digestion and PCR identification of the Recombinantrevealed that, VSV-G gene segment was inserted into the vector correctly; Transfectthe pEF1α-VSV-G into BHK-21cell, red fluorescence can be seen under thefluorescence microscope after24hous; Western blot was used to test the VSV-Gprotein in cell total protein, an expected band can be seen in the result, whichindicated that VSV-G protein expressed successfully in the cells. Meanwhile, we have inserted LGALS1gene into pEGFP-N1vector toconstruct recombinant pN1-LGALS1; The result of double enzyme digestion andPCR identification of the Recombinant demonstrated that, LGALS1gene segmentwas inserted into the vector correctly; Obvious green fluorescence can be seen24hours after the transfection of pEGFP-N1into BHK-21cells under the fluorescencemicroscope; Western blot test showed a higher expression of LGALS1in cells thatwere transfected with pN1-LGALS1than which in cells that were transfected withpEGFP-N1, indicated that LGALS1protein expressed successfully in the cells.Then, we located the interaction of VSV-G with LGALS1by indirectimmunofluorescence observation, yellow fluorescence is visible as the red andgreen fluorescence overlapped each other, which primitively demonstrated theinteraction between VSV-G and LGALS1; The result ofco-immunoprecipitation(Co-IP) showed that, the combination of VSV-G andLGALS1can be screened by rabbit anti-VSV-G, yet LGALS1alone cannot bescreened by the same antibody, which further verified the interaction betweenVSV-G and LGALS1; The MDBK cells were infected with VSV combined withdifferent quality of LGALS1, and exerted less obvious CPE as VSV were combinedwith more LGALS1, which indicated that excessive LGALS1could hinder theinfection of cells by VSV; Infected the MDBK cells with certain amount of VSV anddetected the change in expression of LGALS1, the result showed significant increasein expression of LGALS1in cells after VSV infection; To sum up, we can ultimatelysay that VSV-G protein can inteact with LGALS1.The study not only lay the foundation of in-depth exploration of VSpathogenesis, which helps to reveal the virus infection and replication process andsome other molecular pathogenic mechanism, but also provide material basis for thedevelopment of new prevention and treatment of preparations with the receptorprotein.