Cloning, Expression And Antiviral Activity Against FMDV Of Sus Scrofa/Bos Taurus Interferon Induced Transmembrane Protein Genes

Interferon inducible transmembrane(IFITM) proteins belong to a larger family of membrane-associated proteins—the CD225 protein superfamily,including two hydrophobic membrane-associated domain(IM1, IM2) divided by a conserved intracellular cyclic structure.On the basis of expression of endogenous, the induction of interferon and infection of virus will further upset the expression.IFITMs hold diversified functions,involve embryonic development, cell adhesion/proliferation and tumor formation. Recent years, the significant effects in antiviral aspect make IFITM become the hotspot again—By the current, IFITMs antiviral spectrum has contains at least 20 kinds of viruses involved in 10 families(including Orthomyxoviridae, Retroviridae, Togaviridae, Coronaviridae, Filoviridae, Flaviviridae, Rhabdoviridae, Hepadnaviridae, Reoviridae) since anti-viral activities to IAV induced by IFITMs were indentified in 2009 reported by Cell. In this regard, human and mouse IFITMs are always the key points in researches,but in the field of other species, there are’t enough analyses. In view of the anti-viral research results of human IFITM1-3 and mouse IFITM3, sus scrofa/bos taurus IFITM1-3 cluster may be equipped with the same effect. At present, it has been reported about sus scrofa IFITM3 restrict the replication of IAV, IFITM1, IFITM 2 and bos taurus IFITMs don’t have much study.FMD is the most serious epidemic to pigs, cattle livestock husbandry. As it will degrade the quality and quantity of animal products to affect the commodity circulation internationally, resulting in huge economic losses and other reasons, the OIE listed it as the animal epidemic disease that must be reported. Considering the importance that pigs, cattle livestock husbandry promote agricultural development and the great proportion taken by pork and beef among the meat consumption in all over the world, the impact that the outbreak of epidemic will be very bad. Meanwhile, the antiviral spectrums of IFITM are more concentrated in enveloped viruses, nonenveloped viruses except reoviruses don’t have many reports. sus scrofa/bos taurus IFITMs limiting non-enveloped virus FMDV replication has two significances: on one hand, it perfect relevant information about IFITMs in heavy livestock, on the other hand, also expand the anti-viral spectrum of IFITM again.We acquire sus scrofa or bos taurus IFITMs(s/bIFITMs) coding sequence from pig PBMC cell, PK-15, MDBK and these IFITM genes were subcloned to pMD18-T simple vectors. After sequencing, to build s/bIFITMs relevant biological informations by various bioinformatics softwares. Meanwhile, the target genes were inserted respectively into the eukaryotic expression vector pLV-EGFP, resulting into pLVsIFITM1, pLV-sIFITM2, pLV-sIFITM3, pLV-bIFITM1, pLV-bIFITM2, pLVbIFITM3.Then these recombinant plasmids were transfected into HEK293 T, BHK-21.The expression of target genes were detected by RT-PCR, Western blot.To explore target proteins antiviral activities against FMDV, we choose susceptible cell BHK-21 as transfection target cell. By lipo2000 lipidosome to transfect recombinant plasmid to BHK-21, to construct BHK-21 cell lines that stable overexpress sus scrofa/bos taurus IFITMs, and control cell lines and identified by western blot, named for BHK-sIFITM2, BHK-sIFITM3, BHK-bIFITM2, BHKbIFITM3, BHK-puro. As for some unknow reasons, we don’t get the corresponding cell lines that overexpress sIFITM1, bIFITM1. In anti-viral aspect, after infected FMDV, using microscopic observation, plaques analysis and real-time quantitative reverse transcription-PCR to demonstrate the anti-viral functions of target proteins to FMDV, providing experimental foundations for further exploring the antiviral mechanism of s/bIFITMs and animal transgenic antiviral breeding.