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 2.2×10⁸ cfu/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