P58IPK论文猪P58(IPK)基因的克隆表达及甲型流感病毒感染对其表达的影响.doc

P58IPK论文：猪P58(IPK)基因的克隆表达及甲型流感病毒感染对其表达的影响 【中文摘要】P58IPK是干扰素诱导、dsRNA激活的蛋白激酶PKR的胞内抑制剂。流感病毒感染后P58IPK在翻译后水平激活,P58IPK的激活导致PKR介导的eIF-2α磷酸化水平降低,使病毒蛋白表达水平增高,对病毒复制有利。然而,敲除P58IPK的小鼠感染病毒后死亡率却显著升高,并有严重的肺部病变和炎症反应。本研究旨在分析猪P58IPK在流感病毒感染中的作用,具体实验内容与结果如下:1.本研究依照人P58IPK基因序列克隆得到猪源P58IPK基因,并用生物信息学分析猪源P58IPK的序列特征与进化关系。猪源P58IPK全长1 518 bp,编码505个氨基酸,蛋白的N端和C端高度保守,猪源P58IPK与人源P58IPK蛋白相似度高达99.21%。2.将P58IPK克隆到pET-32a原核表达载体上,转化到大肠杆菌表达菌BL21（DE3）中,在IPTG浓度为0.2 mmol/L,37℃诱导7 h时,融合蛋白His-P58IPK可溶性表达量最大,经HiTrapTM Chelating HP层析柱纯化后免疫新西兰大白兔,获得P58IPK的多克隆抗体。经检测多抗效价达到1:100000,具有特异反应性。3.将基因克隆到pEGFP-C1和pEGFP-N1真核表达载体上,采用脂质体转染法转染猪脐静脉血管内皮细胞（SUVECs）。在转染后24 h时融合蛋白EGFP-P58IPK-C1和P58IPK-EGFP-N1荧光表达量最大。根据荧光发光位置判定猪源P58IPK蛋白定位于细胞质,48 h后收集细胞进行裂解,Western blotting检测到融合蛋白EGFP-P58IPK和内源性P58IPK蛋白。4.甲型流感病毒株H1N1和H3N2分别感染健康猪,7 d后取猪肺脏组织,一部分提RNA并逆转录为cDNA,采用实时定量PCR方法检测P58IPK基因的变化,另一部分保存在4%的甲醛溶液中,做肺部病理组织学切片和P58IPK蛋白的免疫组织化学检测。结果表明感染病毒的猪肺脏组织发生明显病理变化,机体P58IPK的mRNA表达量和蛋白表达量在病毒感染后均上调。 【英文摘要】P58IPK is a cellular inhibitor of PKR that is activated at the post-translational level in response to influenza A virus infection. The activation of P58IPK results in reduced level of PKR-mediated eIF2αphosphorylation, which has long been thought to benefit influenza virus by maintaining a high rate of viral protein translation. However, it was found that influenza A virus infection was more lethal in mice lacking P58IPK, due to increased lung pathology and inflammation. In order to confirm the function of porcine P58IPK in influenza A virus infection and further explore the mechanisms, we made the experiments as follows:1. The swine gene P58IPK was cloned through homology searching in the swine EST database. Then this gene was cloned using reverse transcription polymerase chain reaction （RT-PCR）. The cDNA of the gene contains the complete open reading frame （ORF） of 1518 bp, encoding 505 amino acid residues. The gene was preliminarily analyzed using bioinformatics methods. The N-terminal and C-terminal were highly conservative, and the similarit