中文摘要：

【目的】构建肺组织细胞Calu-3及A549的高质量酵母cDNA文库并筛选与流感病毒NP蛋白相互作用的宿主因子,为深入研究流感病毒NP蛋白功能、病毒复制及致病机制奠定基础。【方法】提取等量Calu-3及A549细胞的总RNA,混合后反转录生成cDNA,利用长距离PCR（LD-PCR）扩增合成dscDNA,用CHROMA SPINTM＋TE-400纯化柱纯化dsDNA,按照Clontech公司的Make Your Own＂Mate＆Plate＂Library System操作程序,将带有同源臂的dscDNA与线性化p GADT7-Rec共同转化Y187酵母感受态细胞,涂布SD/-Leu平板后于30℃培养4d左右,收集所有菌落,混匀分装即为Calu-3和A549细胞cDNA的酵母文库,并对文库库容、滴度及多样性进行分析。利用Eco RⅠ和Bam HⅠ双酶切将A/Auhui/2/2005（H5N1）NP定向插入pGBKT7载体,构建高致病性流感病毒NP的诱饵质粒p GBKT7-NP,经验证该质粒无自激活活性,并进一步采用构建完成的Calu-3/A549细胞酵母文库进行杂交筛选,筛选得到的正确阅读的猎物质粒与诱饵质粒共转Y2H Gold酵母菌,分别以BD-P53/AD-T7作为阳性对照和BD-Lam/AD-T7作为阴性对照,挑取最终在SD/-Trp/-Leu/-Ade/-His/X-α-gal/Aro A（SD/-4/X/A）固体培养板上生长良好且变蓝的菌落,即为候选与目标蛋白互作阳性的蛋白,提取酵母质粒,进行测序分析、序列比对和Gene Ontology分析。【结果】提取两种细胞RNA 28S与18S条带清晰,5S条带暗淡,表明所提RNA质量较高,基本无降解;对提取的RNA反转录纯化获得dscDNA,dscDNA条带呈弥散状,片段大小分布于500—2 000bp之间,说明不同丰度及大小的RNA均成功反转录;构建的dscDNA文库库容为1.5×10^7,滴度为2.2×10^8cfu/m L,重组率为88%,PCR鉴定文库插入片段,条带大小不一、多样性好;利用诱饵质粒与文库进行双杂交筛选,回交验证后得到11个与NP蛋白互作的宿主因子。经Gene Ontology分析显示,11个宿主因子参与的生物过程包括：细胞凋亡、胚胎发育、可变剪?

英文摘要：

【Objective】In order to study the biological function of influenza virus nucleoprotein and provide a basis for the understanding of mechanism of influenza virus replication and pathogenesis, a yeast two-hybrid library derived from human lung epithelial cell lines Calu-3and A549 was constructed, and the host factors that interact with nucleoprotein（NP） of influenza virus were screened. 【Method】Total RNA was extracted from equal numbers of Calu-3 and A549 cells and their c DNA was synthesized by reverse transcription. Double strand c DNA（dsc DNA） was amplified by long-distance PCR（LD-PCR） and purified through CHROMA SPINTM＋TE-400 column according to the user manual of Make Your Own ＂Mate Plate＂ Library System（Clontech）. The purified dsDNA containing homologous arms was transformed into component Y187 yeast cells together with linearized p GADT7-Rec plasmid, and then the samples were incubated on SD/-Leu plates at 30℃ for about 4 days. All colonies were harvested and aliquoted, resulting in the construction of yeast two-hybrid library of Calu-3 and A549 cells. The library quality was evaluated by capacity, titer, recombination efficiency and diversity. Meanwhile, the Eco RI and Bam HI digested NP gene from influenza virus A/Anhui/2/2005（H5N1） was inserted into p GBKT7 vector to generate the bait plasmid, which was further demonstrated without self-activating activity. The bait plasmid p GBKT7-NP was transformed into Y2H-Gold yeast strain, and then used to screen for interacting proteins from the yeast two-hybrid library. The selected pray plasmids with correct encoding insert and bait plasmids were co-transformed into the yeast cells, with BD-P53/AD-T7 and BD-Lam/AD-T7 plasmids as positive and negative controls, respectively. The blue colonies that grew well in medium containing SD/-Trp/-Leu/-Ade/-His/X-α-gal/Aro A（SD/-4/X/A） were selected as candidate containing potential interacting partners of NP. After plasmid extraction and sequencing analysis, these candidates were annot