Screening And Preliminary Identification Of Receptor Of Vesicular Stomatitis Virus G Protein

Vesicular stomatitis(VS) is a high contagious zoonosis disease which is causedby vesicular stomatitis virus (VSV), the clinical symptoms of VS closely resemblesfoot-and-mouth disease, swine vesicular disease, and vesicular exanthema of swine,and normally it is difficult to distinguish between them. The clinical characteristics ofthe disease are vesicles, ulcers and scab on the lips, hoof crown, and udder. Painwhich was caused by ulcers can lead to lose animal’s appetite and secondary mastitis,thus it results in decreased productivity in the species, and even death, and severeeconomical losing to aquaculture. VSV also infects human and causes flu-likesymptoms. Thus, it is significant for prevention and treatment of VS and for the healthof human to reveal about molecular pathogenic mechanism of VSV. Given the widevariety of infected cells and animals by VSV, and glycoprotein (G) which is one ofthe main structure proteins of VSV can be combined with surface receptor of the hostcell to cause membrane fusion, and has played a key role in the process of virusinfection, but there is no consistent understanding of the receptor. In addition, yeasttwo hybrid system was established by Fields and Song when they studied eukaryoticgene transcriptional regulation in1989. In recent years, the technology is widely usedin the study of interaction between protein-protein or finding new genes, cell signaling,tumor gene expression and drug screening, and other fields. It is a simple and efficientmolecular biology technique. Therefore, from the view of cell receptors, this researchused yeast two hybrid system to screen receptors of VSV-G protein in MDBK cell andpreliminarily identified them. It provided new thoughts for VS control and preventionand brought new reference value for the molecular pathogenesis of VSV.Firstly, in this study we constructed cDNA library of MDBK cells by using MakeYour Own“Mate&Plate” Library System kit of Clontech company. The cDNAlibrary of MDBK cells was analysed by the quality evaluation. The library transform efficiency of cDNA library was2.6x106. The cell density was1.7x108/mL.Thelibrary titering was5.6x107cfu/mL. Most of the inserted fragments length werebetween0.5kb to3.0kb, and the average length was about1.1kb. It suggested thatthe constructed cDNA library was better quality and could be used for screening ofyeast two hybrid system.Secondly, this study choosed VSV-G protein as the bait to fish for the host cellreceptor from cDNA library which interacted with VSV-G. According to the design ofa pair of specific primers, RT-PCR amplified VSV-G protein genes, and the fragmentsize was about1536bp. By the analysis, the purpose gene correctly inserted intopGBKT7bait vector. Test analysis of activation and toxicity about the bait proteinshowed that the bait protein did not produce the activation and toxic effect. Itsuggested that we builded expression vector (pGBKT7VSV-G) of the bait could beused as a bait protein to screen cDNA library of MDBK cells.Finally, library strains Y187(pGADT7-Rec-AD/library) with bait strains Y2H(pGBKT7-VSV-G) were hybridized for screening. After two screening of the DDO/Xand QDO/X/A selective mediums, we got five candidate positive clones. Sequencedcomparative analysis showed that they were lectin galactoside-binding, soluble,1(LGALS1) and ribosomal protein S20(RPS20). In order to further determine theinteractions between two proteins and bait protein, this study verified again that theLGALS1and RPS20proteins could interact with bait protein (VSV-G) through themutual confirmation test and His pull-down test, but the interactions between RPS20and VSV-G was relatively weak and had poor specificity. The study not only lays thefoundation for researching the roles of the G protein and its receptors in thepathogenic molecular mechanism of VSV, at the same time helps to reveal a series ofscientific problems about the virus infection and replication process, but also providsimportant theoretical basis for really effective prevention and control of the diseaseand breeding for disease resistance.