The Study Of Lentiviral-mediated RNA Interference Targeting The Gene Of Bovine Integrin αv Subnuit

Viral receptor plays a significant role in the interaction between virus and its cellular receptors. The attachment of foot-and-mouth-disease virus to the receptor on the surface of host cells is the first stage of virus infection, through which virus can entry the cell. Then virus completes the replication of its genome and the assembly of the viral particles. Integrins are a large family of heterodimeric transmembrane glycoproteins composed ofαandβsubmint that interact noncovalently at th cell surface. They mediate cell-cell interaction and the binding of cells to the extracellular matrix,and they play a crucial role in cell division, differentiation, migration, and survival. The protein family has 24 members. But onlyανβ1,ανβ3,ανβ6,ανβ8 four members have been identified as receptors for foot-and-mouth disease virus at present, and they have the same subunitαν. RNA interference refers to the inhibition of gene expression by dsRNA. It is a post transcriptional gene silencing mechanism, by which double stranded RNA specifically suppresses the expression of coherent sequences. As a good eukaryotic cells gene transfer vector, lentivirus-based vectors are generally applied in RNAi related research, such as antiviral research, therapy of hereditary diseases and gene therapy. Considering lentiviral vector can stably transfect host cells and even integrate into the host genomes, it can be used to express the designed siRNA in host cell. Thus continuously downregulating the express of the target gene.According to the sequence of bovine integrin published in Genbank, we designed three siRNAs targeting integrinανsubunit by the online software of designing siRNA namedαν-2270,αν-1716,αν-483. Moreover, a negative control named si0 is also made. The most efficient siRNA(αν-483) was selected by real-time RT-PCR. Authorized a biotechnology company to synthetize the corresponding ds oligo cDNA sequence ofαν-483 and the cloned the ds oligo into the U6 entry vector, thus construst the entry vector U6-αν483. After the LR recombination reaction between the entry vector U6-αν483 and the expression vector in the pLenti6/BLOCK-iT^TM system, the sequence of the U6 RNAi cassette in U6-αν483 has been transferred into the destination vector and the expression vector named DEST-αν483 was constructed. Transfected the DEST-αν483 and the three packaging plasmids pLP1,pLP2 and pLP/VSVG in the packaging mix into well-grown 293FT and harvested virus(named plenti6/αv483)-containing supernatants 72 hours posttransfection. After Titering the plenti6/αv483 lentiviral stock using pk-15 cell line, we figured the titer of the lentiviral construct out: 14.2×10^-6 TU/ml. Then MDBK cells were infected by plenti6/αv483 and were continually seeded in the medium at the optimal blasticidin regulatory concentration for two weeks to obtain the blasticidin resistant colonies. At last, four resistant cells lines named 1B8,1F5,2B9,2E6 were selected and then cultured for positive identification. After the identification by real-time RT-PCR in the nucleic acid level and the cell-ELISA,IIF in the protein level, we confirmed that the expression of integrin in these four cells lines all had different degree of decline. Furthermore, the downregulating expression of integrin in 1F5 cell line is the most significant.The experiment had constructed the lentiviral vector which express the shRNA targeting the bovine integrinανsubunit successfully and then obtained blasticidin resistant cells whose genome had been integrated by the lentiviral vector. We had carried out the preliminary study of anti-virus by downregulating the expression of the viral receptor.