【作者】 王蒙；

【导师】 刘红林；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2008， 硕士

【摘要】 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PolⅢH1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TCID₍（50）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。更多还原

【Abstract】 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 PolⅢH1 promoter（pSUPER-H1）.Three pairs of oligonucleotides comprised of 64 bases were chemically synthetized and annealed,pSUPER-H1 vectors were linearized with BglⅡand HindⅢ.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.更多还原