SiRNA Targets To FMDV Receptor Integrin β6Subunit To Inhibit Virus Replication

Object:Integrin is a cell receptors mediated virus invades host cells, play an important role in the virus adhesion process. In this study, cytoplasmic domain of FMDV receptor integrin β6subunit was modified using RNA interference technology to suppress β6gene expression, in order to inhibit virus replication in host cell. Our research is to establish the basis to successfully obtain the anti-Foot-and-Mouth Disease transgenic pigs.Methods:We designed seven pairs of siRNA interference molecules base on the porcine integrin β6subunit gene. siRNA that interfered with β6receptor gene were selected and synthesized shRNA. Seven interference vectors were constructed and those were confirmed were applied to extract plasmid, purified and concentrated by ethanol precipitation. Using tissue and enzymatic digestion method, porcine embryo fibroblast was separated from Pietrain which has been pregnant for32days, and cultured in20%FBS DMEM Medium in5%CO2incubator. Interference plasmids were transfected into porcine embryo fibroblast and BHK cells using X-tremeGENE HP DNA transfection Kit. Green fluorescence protein expression of interference plasmid in PEF cells was assayed by fluorescence convert microscope. Effects of interference vectors were verified by real-time quantitative PCR. The more effective vectors were digested by restriction enzyme and linked with integration vector PXL-EGFP-NEO. Plasmids were extracted and transfected PEF cells after purification and concentration by ethanol. We screened these transfected cells by G418to obtain a purified PEF cells and expanded reproduction. qRT-PCR was used to detect the suppression effect of integration vectors and quantity of virus replication. Virus attack test has been carried out.Results:Porcine integrin β6subunit gene sequence was searched in NCBI, and its accession ID is EF432729. We compared this sequence with mouse integrin β6subunit sequence by DNAMAN and found84.75%homology, as well as93.55%homology in target domain. We designed and synthesized seven siRNA, constructed interference vectors and named them respectively. Plasmids were extracted from interference vectors that were confirmed by sequencing, purified and concentrated to over3000ng/μL.72tubes of PEF cells F1generation were liquid nitrogen cryopreservation. The interference plasmids were transfected to PEF cells F1generation, and relative high transfection efficiency was achieved through the optimization of transfection conditions. RNA of interfered PEF cells was extracted and reverse transcripted into cDNA. The expression of porcine integrin β6subunit inhabited by interference plasmids was determined by qRT-PCR. The more effective interference plasmid pGsi-Z4was selected and its efficiency is91.7%. The more effective integration vectors were constructed and screened by G418after transfected PEF cells based on Neo resistance of integration vector PXL-EGFP-NEO then expanded reproduction. A better interference effect of integration vector was examined by qRT-PCR in36hours after transfected. Quantity of virus replication was decreased in24hours and36hours after virus attack, which also detected by qRT-PCR.Conclusion:We designed siRNA based on cytoplasmic domain of FMDV receptor integrin β6subunit, and a better interference effect plasmid pGsi-Z4in PEF cells was obtained. This plasmid can effectively inhabit the expression of receptor subunit, reduce the quantity of virus invaded cells, and play an important role in inhabiting virus mass replication in cells. This effect was not ideal using pGsi-Z4for suppressing P6gene expression in BHK cells because it had a base pair mismatch with siRNA target site. pGsi-Z5was completely match with siRNA target site, can work with inhibition in some degree. The better interference effect of integration vector appeared in36hours after transfection. The relatively better results of reducing the virus replication were shown in24hours and36hours after transfection.