人源肺细胞cDNA文库构建及与流感病毒NP互作宿主蛋白的筛选

doi:10.3864/j.issn.0578-1752.2016.22.017

摘要/Abstract

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pGADT7-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EcoRⅠ和BamHⅠ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pGBKT7-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/AroA(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1.5×10⁷，滴度为2.2×10⁸cfu/mL，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、可变剪接、转录调节及细胞增殖等；涉及的分子功能包括：GTP结合活性、金属离子结合活性、DNA结合活性及转录因子活性。【结论】成功构建同时含有Calu-3和A549两种人源肺细胞cDNA的酵母文库，文库覆盖cDNA更全面，为后期筛选与流感病毒其它蛋白互作的宿主蛋白奠定基础，筛选得到与NP蛋白存在相互作用的宿主因子为进一步深入研究NP功能提供了可靠的前期数据。

关键词: 流感病毒, NP蛋白, cDNA文库构建, 酵母双杂交系统, 宿主因子

Abstract: 【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 cDNA was synthesized by reverse transcription. Double strand cDNA (dscDNA) 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 pGADT7-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 EcoRI and BamHI digested NP gene from influenza virus A/Anhui/2/2005 (H5N1) was inserted into pGBKT7 vector to generate the bait plasmid, which was further demonstrated without self-activating activity. The bait plasmid pGBKT7-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/AroA(SD/-4/X/A) were selected as candidate containing potential interacting partners of NP. After plasmid extraction and sequencing analysis, these candidates were annotated and classified by Blast and Gene Ontology (GO) analyses. 【Result】Analysis of RNA extracted from the two types of cells showed that both 28S and 18S RNA bands were clear whereas 5S band was faint, indicating that high quality RNA was obtained with almost no degradation. The dscDNA reverse transcribed from RNA was evenly dispersed on the gel with sizes ranged mostly between 500 and 2000 bp, demonstrating that RNA with different abundance and sizes were successfully reverse transcribed. The capacity and titer of the established yeast two-hybrid library were 1.5×10⁷ and 2.2×10⁸cfu/mL, respectively, with 88% recombination efficiency and sufficient diversity. The screening of the library with the NP bait revealed 11 candidate interacting proteins. Through gene ontology analysis, it was found that these proteins are involved in several biological processes including regulation of cell apoptosis, embryonic development, alternative splicing, transcription regulation translation, metabolic process, regulation of cell proliferation etc. In addition, the molecular functions of these proteins included GTP binding, metal ion binding, DNA binding and transcription factor activity.【Conclusion】 In conclusion, the yeast two-hybrid library containing Calu-3 and A549 cells was successfully constructed, which laid a foundation for the screening of host factors interacting with other influenza virus proteins, and the identification of 11 potential interacting host factors provided preliminary data for further study of the biological function of influenza virus NP protein.

Key words: influenza virus, NP protein, cDNA library construction, yeast two-hybrid (Y2H) system, host factor

罗维玉，朱鹏阳，张杰，胡永浩，孔晖晖，梁立滨，周圆，李呈军，姜丽，陈化兰. 人源肺细胞cDNA文库构建及与流感病毒NP互作宿主蛋白的筛选[J]. 中国农业科学, 2016, 49(22): 4451-4459.

LUO Wei-yu, ZHU Peng-yang, ZHANG Jie, HU Yong-hao, KONG Hui-hui, LIANG Li-bin, ZHOU Yuan, LI Cheng-jun, JIANG Li, CHEN Hua-lan. Construction of cDNA Library Derived from Human Lung Epithelial Cell Lines and Screening for Host Cellular Proteins Interacting with Influenza Virus Nucleoprotein[J]. Scientia Agricultura Sinica, 2016, 49(22): 4451-4459.

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