本实验采用RNA干涉（RNAi）的方法开展抗禽流感病毒转基因鸡的研究，首先将禽流感病毒H9N2毒株核蛋白基因（NP）插入真核表达载体pL-EGFP中EGFP的上游，获得了NP-EGFP融合基因表达载体pL-NP-EGFP作为禽流感病毒siRNA筛选质粒，继而针对AIV各亚型核蛋白基因（NP）的保守序列，设计合成三对shRNA，并退火接入带有Pol III H1启动子的干涉质粒（pSUPER-basic-H1），分别命名为：pSUPER-siNP1、pSUPER-siNP2、pSUPER-siNP3。重组质粒经双酶切鉴定并测序确认。将此干涉质粒与NP基因筛选质粒pL-NP-EGFP（NP/EGFP infusion gene）共转染鸡胚成纤维细胞，较其他两个干涉质粒，pSUPER-siNP3高效抑制了NP基因的表达，为进一步确认比较此重组质粒对AIV的抑制，本实验进行了禽流感hemagglutination ( HA)滴度和TCID50测定，重复三次均获得相似结果，pSUPER-siNP3转染组H9N2 的TCID50分别降为：10-5.00/0.1 mL;10-5.16/0.1mL;10-5.28/0.1mL；HA效价降为：1：4。而未转染干涉质粒禽流感病毒对照组TCID50为：10-8.50/0.1 mL,HA效价为：1:256。利用该高效干涉片段本实验构建了pLenti6-siNP3-EGFP慢病毒表达载体, 并进一步确认比较了慢病毒表达质粒的干涉效果：pL-NPi-EGFP转染组H9N2的TCID50降为：10-5.16/0.1 mL，HA效价降为：1：4；而未转染干涉质粒禽流感病毒对照组H9N2的TCID50为：10-8.33/0.1 mL，HA效价为：1：256。最后将慢病毒表达载体与辅助质粒共转染293 FT 细胞生产病毒颗粒。将病毒浓缩后胚下腔显微注射转染鸡胚，孵化率达10%（12/120），结果在检测的12只受体鸡胚胎中，有4只发生嵌合。

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 PolIII H1 promoter (pSUPER-H1) . Three pairs of oligonucleotides comprised of 64 bases were chemically synthetized and annealed . pSUPER-H1 vectors were linearized with Bgl IIand Hind III. 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.