P58IPK论文猪P58(IPK)基因的克隆表达及甲型流感病毒感.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-2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 eIF2phosphorylation, 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 similarity of P58IPK protein between swine and human was 99.21%.2. P58IPK was cloned into a prokaryotic expression vector pET-32a to construct a recombinant plasmid pET-P58IPK. The fusion protein His-P58IPK was expressed in E.coli BL21 and purified using a His-tag protein purification column. Subsequently, New Zealand white rabbits were immunized with the purified protein. Specific polyclonal antibodies against the fusion protein His-P58IPK was obtained. The titer of the antibody was 1: 100000 detected by ELISA. The protein p58IPK could be specifically detected by Western blotting and immunofluorescence assay using the polyclonal antibodies.3. We inserted porcine P58IPK into eukaryotic expression vetor pEGFP-C1 and pEGFP-N1, which were named as EGFP-P58IPK-C1 and P58IPK-EGFP-N1, respectively. Each recombinant plasmid containing a green fluorescence reporter gene was transfected into SUVECs （Swine Umbilical Vein Endothelial Cells） by lipofectamine-2000. Fluorescence microscope showed that fusion protein EGFP-P58IPK-C1 and P58IPK-EGFP-N1 were at the best expression level 24 hours post transfection. According to the fluorescence position of the cells, we concluded that porcine P58IPK was localized to the cytoplasm. Western blotting analysis confirmed the expression of P58IPK in vitro and in vivo. 4. Healthy piglets were infected with H1N1 and H3N2 influenza A virus strains, respectively. Seven days later, lung tissues were collected from the healthy controls and infected piglets, respectively. RNA was extracted from a part of lung tissue samples and reversely transcripted for qRT-PCR detection. Another part of lung tissue samples were made for pathological slices and P58IPK immunohistochemical assay. Pathological changes were observed on the slices. These results showed that the expression of porcine P58IPK in the levels of mRNA and protein were up-regulated in infected piglets.【关键词】P58IPK 多克隆抗体 Western blotting qRT-PCR 免疫组化【英文关键词】P58IPK Polyclonal antibody Western blotting qRT-PCR Immunohistochemistry【目录】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摘要6-7ABSTRACT7-8论文综述13-26第一章 哺乳动物P58(IPK)基因的研究进展13-261.1 DnaJ 基因家族研究进展13-141.1.1 DnaJ 基因家族的成员及命名131.1.2 DnaJ 基因家族的功能和作用机制13-141.2 DnaJ 家族蛋白P58(IPK)研究进展14-171.2.1 DnaJ 家族蛋白P58(IPK)的发现与结构特点141.2.2 DnaJ 家族蛋白P58(IPK)的TPR 结构14-161.2.3 DnaJ 家族蛋白P58(IPK)的DnaJ domain16-171.3 P58(IPK)病毒感染过程中宿主防御的细胞抑制剂“Cellular Inhibitors of the Host Defense”（CIHDs）17-191.3.1 P58(IPK)对病毒复制的贡献17-181.3.2 P58(IPK)在病毒感染过程中对宿主的贡献18-191.4 P58(IPK)抑制PKR 介导的细胞凋亡19-201.5 P58(IPK)基因在内质网应激反应中下调eIF-220-211.6 P58(IPK)基因在内质网应激中抑制PERK211.7 P58(IPK)基因调控未折叠蛋白应答（UPR）21-221.8 P58(IPK)在内质网应激中发挥保护作用22-231.9 P58(IPK)在柯萨基病毒（CV83）感染后UPR 作用网络中下调23-241.10 P58(IPK)基因在植物中存在同源物241.11 P58(IPK)蛋白的广泛表达24-26研究内容26-67第二章 猪P58(IPK)基因的克隆与序列分析26-382.1 材料26-272.1.1 样品来源26-272.1.2 工程菌种和质粒来源272.1.3 试剂和设备272.1.4 分子生物学分析软件和数据库272.2 方法27-322.2.1 电子克隆猪P58(IPK)27-282.2.2 分子克隆猪P58(IPK)28-302.2.3 阳性重组质粒菌液PCR 鉴定和测序30-322.3 结果与分析32-372.3.1 电子克隆猪P58(IPK)结果322.3.2 PCR 产物P58(IPK)电泳结果322.3.3 重组质粒pMD-P58(IPK)菌液PCR 电泳结果32-332.3.4 pMD-P58(IPK)测序结果与序列分析33-342.3.5 猪源P58(IPK)外显子软件预测结果342.3.6 猪P58(IPK)蛋白三级结构预测34-352.3.7 P58(IPK)进化分析35-362.3.8 猪P58(IPK)蛋白与其它物种蛋白的相似性分析结果36-372.4 讨论372.5 小结37-38第三章 猪P58(IPK)基因的原核表达、蛋白纯化与多抗制备38-503.1 材料38-393.1.1 试剂和酶383.1.2 工程菌和质粒383.1.3 仪器设备38-393.2 方法39-443.2.1 pET-P58(IPK)重组载体的构建与鉴定39-403.2.2 pET-P58(IPK)重组质粒转化表达菌 BL21 及鉴定403.2.3 pET-P58(IPK)重组质粒阳性菌BL21 诱导表达403.2.4 SDS-PAGE 检测融合蛋白His-P58(IPK)表达40-413.2.5 His-P58(IPK)融合蛋白的纯化41-423.2.6 P58(IPK)融合蛋白抗血清制备423.2.7 P58(IPK)多抗效价检测42-433.2.8 Western Blotting 检测His-P58(IPK)融合蛋白的表达433.2.9 免疫荧光检测P58(IPK)多抗对天然抗原的识别43-443.3 结果与分析44-483.3.1 重组质粒pET-P58(IPK)酶切鉴定结果443.3.2 IPTG 诱导His-P58(IPK)融合蛋白表达SDS-PAGE 分析结果44-453.3.3 His-P58(IPK)重组蛋白纯化后SDS-PAGE 分析结果45-463.3.4 多抗效价检测结果46-473.3.5 Western Blotting 检测His-P58(IPK)融合蛋白表达的结果473.3.6 免疫荧光检测P58(IPK)多抗对天然抗原的识别结果47-483.4 讨论483.5 小结48-50第四章 猪P58(IPK)基因在SUVEC 细胞中表达50-584.1 材料504.1.1 载体、细胞和菌种504.1.2 酶和试剂504.1.3 主要仪器504.2 方法50-544.2.1 构建P58(IPK)-pEGFP-C1 和pEGFP-P58(IPK)-N1 重组真核表达载体50-534.2.2 细胞培养和质粒转染534.2.