Study On The Lentiviral-siRNA System Targeted To Aiv And Transgenic Chickens Production

To construct expressing vector of shRNA in order to inhibit Influenza virus production.First pL-NP-EGFP,an enkaryotic expression vector containing NP-EGFP,was constructed by inserting NP gene of AIV strain into upstream of EGFP gene in eukaryotic expression vector pL-EGFP.Then targeting NP we constructed the plasmid containing Polâ…¢H1 promoter(pSUPER-H1).Three pairs of oligonucleotides comprised of 64 bases were chemically synthetized and annealed,pSUPER-H1 vectors were linearized with Bglâ…¡and Hindâ…¢.The annealed oligonucleotides were inserted into downstream of H1 promoter to construct the recombinant shRNA plasmid(pSUPER-H1-siNP).The recombinant plasmids were identified by enzyme digestion.and sequence analysis.To detect the effect of inhibition,we cotransfect the recombinant plasmid and the screened plasmid pL-NP-EGFP(NP/EGFP infusion gene).The activity of the fluorescence in chicken embryo fibroblastic(CEF) cells transfected with the plasmid pSUPER-H1-siNP3 was lower than others and the nontransfectedcells,then It is confirmed by the hemagglutination(HA) titre and TCID₅₀ of Influenza virus.The H9N2 TCID50 is 10^5.00/0.1 mL,HA is 1:4 transfected with pSUPER-siNP3 and the TCID₅₀ of H9N2 control is 10^-8.33/0.1 mL,HA is 1:256.It showed that the recombinant plasmid could specifictly inhibit the virus production in CEF cells,then a lentiviral vector pL-NPi-EGFP was constructed and The H9N2 TCID₅₀ is 10^-5.16/0.1 mL,HA is 1:4 inhibited by pL-NPi-EGFP and the TCID₅₀ of H9N2 control is 10^-8.33/0.1 mL,HA is 1:256.then pLenti6-siNP-EGFP was constructed and harvested the virus by cotransfecting 293 FT cells with the vector and packaging plasmids.Lentiviruses after concentration were used to transfect chicken embryos,A total of 120 eggs were injected with lentivirus in our experiment from which 12(10%) chicken hatched.4 out of 12(33%) chickens were determined to contained vector sequences PCR using EGFP specific primers.