A Preliminary Study On The Production Of Transgenic Sheep By Lentivirus Mediated RNAi Technology

The interference of RNA?RNA interference,RNAi?is a new technology applied to the field of molecular biology.Since RNAi was discovered,RNAi technology has been widely used in animal and plant genetic improvement,functional genomics,gene therapy and resistance breeding in some of the studies and research in the field of antiviral in a major breakthrough,is expected to become a new method for disease resistance breeding.This experiment mainly selected ON strain of O type foot-and-mouth disease virus and Nigeria 75/1 strain of peste des petits ruminants virus,the protein genes of ON foot-and-mouth disease virus 3C,3D,VP1 and and Nigeria 75/1 strain of peste des petits ruminants virus N?F?M were used as the target gene.For each target gene,two pairs of small hairpin RNA?short hairpin RNA,shRNA?primers were designed and synthesized,named VP1-1?VP1-2?3C-1?3C-2?3D-1?3D-2 and N1?N2?F1?F2?M1?M2 respectively.According to the principle of RNAi,6 shRNA recombinant expression plasmids were constructed for each virus.After transfection,the virus was inoculated,and the recombinant plasmids that could efficiently inhibit virus replication were screened by real-time TCID₅₀0 and real-time TCID₅₀.The results showed that six recombinant shRNA expression plasmid construction of ON foot-and-mouth disease virus inhibition rate was between 71.5%⁹3.2%,the inhibitory effects of PLKO.1-VP1-2 and PLKO.1-3D-1 was the most obvious,respectively 93.2%and 90.8%;for the six recombinant shRNA expression plasmid construction of Nigeria 75/1 strain of peste des petits ruminants virus inhibition rate was between 17.4%⁴4.1%,which the inhibitory effect of PLKO.1-N1 and PLKO.1-M1 was the most obvious,respectively 41.8%and 44.1%.The recombinant plasmid that can inhibit virus replication efficiently was prepared by lentivirus preparation and drug screening.A stable cell line was obtained.The inhibitory effect of lentiviral interference vector on viral replication was detected by real-time TCID₅₀and real-time PCR.The results showed that the inhibition efficiency of lentivirus interference vector PLKO.1-VP1-2 and PLKO.1-3D-1 was 88.3%⁹5.49%,and the inhibition efficiency of lentiviral interference vectors PLKO.1-N1 and PLKO.1-M1 was 42.34%and 44.32%respectively.The research results show that,at the cellular level chosen to inhibit ON foot-and-mouth disease virus replication and Nigeria 75/1 strain of peste des petits ruminants virus interference fragment and preparation of lentivirus vector,which lays the foundation for the subsequent anti foot-and-mouth disease virus and peste des petits ruminants virus transgenic sheep breeding.