| RNA interference (RNAi) is a natural process of eukaryotic cells through which endogenous or exogenous double-stranded RNA(dsRNA) directs the specific degradation of homologous mRNA. The phenomenon were discovered in almost all organisms. Its mechanism seems to be a cellular defense mechanism against gene invaders such as viruses and transposons.The key factor to initiate RNAi process is the double-stranded small interfering RNAs (siRNAs) with a length of 19~27bp. With the progression of the research on RNAi, people have found that siRNA could be able to inhibit the replication of many viruses in vivo or in vitro. In recent years, many researchers have performed many investigations on the interference of virus replication, and many advances in this respect have been obtained. It has been used as a new powerful tool for gene-specific therapeutics for viral disease.Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious disease among cloven-hoofed animals and was listed on the top of the List A of animal infectious diseases by the Office International des Epizooties(OIE). FMDV is very easy to mutate and lead to new variants appear continually,many new virus variants result that the traditional vaccine is not able to avoid the breakout and propagation of the disease.The discovery of RNAi mechanism and the advance in the anti-virus research open a new field for the prevention and control of FMD. In this study,we successfully performed RNAi to the replication of FMDV in vitro. The 2B and 3D genes are highly conserved among the different serotypes and hypotype of FMDV. According to the conserved sequences of 2B and 3D genes and the design principles recommended by the Invitrogen Company,five shRNA templates and a control were designed, synthesized, and then cloned into the downstream of the U6 promoter of the shRNA expression vector pEN/U6, resulting in six recombinant plasmids.Firstly, the inhibition capability of the shRNA expressing plasmids targeting to 3D gene was investigated at the cellular level. The recombinant plasmid pEGFP+3D was constructed in the expression vector pEGFP-N1. The shRNA expressing plasmids targeting to 3D gene and pEGFP+3D were co-transfected into PK-15 cells respectively.The analysis revealed that pEN/U6-3D1,pEN/U6-3D2, pEN/U6-3D3 and pEN/U6-3D4 could inhibit 3D expression by fluorescence microscope observation and flow cytometry and real-time RT-PCR. The more effective shRNA expressing plasmids are pEN/U6-3D2 and pEN/U6-3D4.Then,the shRNA expressing recombinant plasmids performed the LR recombination reaction,and generated the expression clones,pEX/U6-3D2,pEX/U6-3D4, pEX/U6-2B and the control, pEX/U6-CON. The above expression clones were transfected into 293FT cells with helper plasmids, lentiviruses were harvested at 50 hours post-transfection. These lentiviruses were transducted into sheep primary fibroblasts and PK-15 cells with Lipofectamine 2000 respectively. At 48 h after transduction, the cells were incubated in standard culture medium including blasticidin. Colonies resistant to blasticidin appeared after cells were cultured in selection medium in two weeks. The surviving cell colonies resistant to blasticidin were obtained and subcultured in 6-well plates and propagated, then the cells were frozen. In order to examine if the gene have integrated into genome of the cells, the genomic DNA of cell clones were used for PCR amplification.Six cell strains expressing the FMDV-specific-shRNAs were obtained and two cell strains expressing the negative shRNA were also obtained. The analysis of inhibition capability of FMDV proliferation by CPE observation,TCID50,IFA and real-time PCR showed that FMDV-specific-shRNAs expressed in these cell strains specifically suppress FMDV proliferation. The infection rate of FMDV in these expressing FMDV-specific-shRNAs cell strains were significantly reduced in contrast to those expressing the negative shRNA cell strains and normal untransgenic cells at 24 hours post-infection.
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