【作者】 柳斌；

【导师】 屈孝初； 李文平； 周晓巍；

【作者基本信息】 湖南农业大学 ， 特种经济动物饲养学， 2007， 硕士

【摘要】 核蛋白(Nucleoprotein)基因是禽流感病毒(AIV)基因组的第5片段。在AIV复制早期，NP与AIV的RNA及多聚酶蛋白共同装配成RNP复合体，通过RNP复合体感染宿主细胞上的某些大分子来影响AIV的转录、复制、装配及转运功能，是AIV感染的重要因素。所以，寻找宿主细胞中与核蛋白密切相关的蛋白研究它们之间的相互作用关系，将对阐明AIV感染的分子机制和防制禽流感具有重大意义。本课题旨在通过制备NP多克隆抗体，利用特异性抗体作为蛋白质研究的有效工具进行NP体内表达和相互作用的研究；同时，利用CytoTrap酵母双杂交系统筛选与NP相互作用的蛋白，以探索流感病毒-宿主细胞相互作用关系。首先，首先从感染了H5N1亚型禽流感病毒的MDCK细胞培养液中收获病毒并提取总RNA，克隆了NP基因全长cDNA片段。其次，构建了原核表达质粒pTIG／H5NP，并在大肠杆菌中大量表达并纯化了NP蛋白，获得了抗原。第三，皮下注射免疫家兔制备多抗血清，制备了抗体亲和层析柱亲和纯化抗体。第四，构建了CytoTrap酵母双杂交pSos／NP诱饵表达质粒；Western blotting验证了融合蛋白hSos-NP能在cdc25H(a)酵母菌中稳定表达。最后，利用CytoTrap酵母双杂交系统，将诱饵质粒pSos／NP和人肺细胞文库质粒共转化酵母细胞，从文库中“钓”出了与NP结合的两个阳性克隆。这两个蛋白分别是聚ADP核糖聚合酶家族成员8(PARP8)和锌指蛋白658(ZNF658)。更多还原

【Abstract】 Nucleoprotein gene is the fifth segment 9f genome of avian influenza virues.Forepart, when AIV replicate, the RNA segments are contained within the virues envelopein association with the nucleoprotein and three subunits of viral polymerase (PA, PB1, andPB2),which together form the ribonucleoprotein (RNP) complex responsible for RNAreplication and transcription.That is the most important factors for virues infection.Therefore, it makes sense to illuminate the molecule mechanism of virues infection and findthe way of preventive from AI by finding the other proteins associated with NP in hostcells, and studying the relation of each-interacting.This task aims at preparing of NP antibody, then using the antibody to study the NPexpressing protein in cells and relation of mutual-interacting.At the same time, using theCytoTrap yeast two-hybrid system to fish NP-interacting protein. This study aim is to questfor relation of mutual-interacting about AIV-host cells.First, the virues RNA was obtained from the supernatant of MDCK cell infected H5N1Subtype avian influenza virues by RT-PCR, and the complete cDNA strands of NP wascloned.Second, prokaryotic expression vector pTIG/H5NP was constructed, then it wastransformed into E.coli BL21(DE3)plysS for highly expression; NP prokaryotic expressionproduction was purificated.Third, the purification of NP protein was injected into rabbit,and serums was collected toprepare NP antibody; at the same time, NP antibody affinity columniation was prepared topurificate antibody.Fourthly, the bait vector of NP (pSos/NP) was constructed, and fuse proteins hSos-NPexpressing in cdc25H (a) yeast stably was confirmed by Western blotting.At last,the bait vector pSos/NP and lung cell library were comtransformed into cdc25H-(a) yeast,and two putative positive clones combined with NP were fished utilizing CytoTrapyeast two-hybrid system. The two positive clones are respective Homo sapiens poly (ADP-ribose) polymerase family, member 8 (PARPS) and Homo sapiens zinc finger protein 658(ZNF658).更多还原