禽流感病毒NP cDNA反义逆转录病毒载体的构建及重组病毒的鉴定

Construction of NP Gene Retrovirus Vector for Avine Influenza Virus and Identification of Recombinant

根据反义ＲＮＡ作用原理，以禽流感病毒（ＡＩＶ）Ｈ１４Ｎ５ ｃＤＮＡ全长及５’端２３５个ｂｐ为目的基因，反向插入反转录病毒载体 ｐＬＸＳＮ中，命名为 ｐＬＸＳＮ－ＮＰ及 ｐＬＸＳＮ－ＮＰ，用脂质体包裹重组质粒转染包装细胞 ＰＡ３１７，Ｇ４１８筛选抗性克性，扩增单个克隆后提取包装细胞上清的病毒ＲＮＡ（ｖＲＮＡ），以巢式ＰＣＲ方法检测，以及用包装细胞上清（重组病毒）感染滴定细胞系ＮＩＨ３Ｔ３，表明已筛选出产毒的的克隆细胞质，得到重组逆转录病毒，为反义ＲＮＡ抑制禽流感病毒复制的实验研究奠定了物质基础。

According to the theory of antisense RNA,we inserted full-long the cDNA of NP gene and 5 and 235bp of AIV H14N5 into retroviral vector pLXSN, named pLXas-NP and pLXas-5 -NP respectively. The retroviral plasmid were transfected into packaging cell line PA317 with lipofectin, then by G418 colony screening method. Firstly the virus RNAs(vRNA)were extracted from the virus-containing super- natant of packaging cell lines, detected the vRNA by Nested-PCR. Secondly infect NIH3T3 with the virus-containing supern...