Construction And Selectiono Of Untranslated Region Random Mutagenesis Gene Library Of H5 / H7 / H9 And N2 / N6 / N9 Subtypes Of Influenza A Virus

Objective: Through bioinformatics methods to analyze the untranslated region(UTR)polymorphisms of the H5/H7/H9 and N2/N6/N9 subtypes of the influenza A virus.Utilizing the RNA polymerase I reporting system,with enchanced green fluorescence protein(EGFP)gene as the reported gene,random mutagenesis gene libraries containing random sites of H5/H7H9 and N2/N6/N9 untranslated regions were constructed.The mutation systemswere selected from the randomized mutagenesis libraries of HA / NA-UTR to analyze the expression of the reporter gene(EGFP)qualitatively and quantitatively to explore the influence of UTR mutations on the HA and NA gene expression of influenza A virus.Methods: 1.Characteristics analysis of influenza virus HA/NA-UTR gene sequences: people in shenzhen were infected with H5N6,and the H7N9 virus and environmental monitoring H9N2 influenza virus were sequenced during the period from 2013 to 2015.The sequencing results were combined with NCBI data to analyze the polymorphism sequence characteristics of H5 / H7 / H9 and N2 / N6 / N9-UTR.The MEGA software was used to construct the phylogenetic tree for each HA / NA-UTR sequence.DNAstar software was used to analyze the mutation sites of HA / NA-UTR sequence to obtain UTR mutation site information in natural evolution process.Artificial mutagenesis of each subtypes of UTRs were designed as N-bases(including A,T,C,G),which were used as primers to construct RNA polymerase I fluorescence reporting system containing UTR random mutations.2.Transformation of RNA polymerase I fluorescence reporting system: The RNA polymerase I component system was constructed by inserting the RNA polymerase I human-derived promoter PIh,mouse-derived terminator t1 and Bsmb I restriction site into the p MD18-T cloning vector without the T7 promoter.A confirmed influenza A virus(A / chicken / Beijing / 0309/2013(H9N2))was wild type,and its HA / NA-UTR sequence and reporter gene EGFP sequence were cloned into the RNA polymerase I component system and transfected into the influenza A virus infected MDCK cells.3.Construction of H5/H7/H9 and N2/N6/N9-UTR random mutagenesis gene libraries: The N-based HA / NA-UTR sequences and EGFP reporter gene sequences were cloned into the RNA polymerase I component system to construct H5 / H7H9 and N2 / N6 / N9-UTR random mutagenesis gene libraries.The gene mutation capacity was calculated and 10 positive clones were randomly selected from each subtype.4.Selection and identification of the reporter gene up-regulated clones: Via sequencing appraisal of HA/NA-UTR RNA polymerase I reporter system transfected to influenza A virus infected MDCK cells,the expression of reporter EGFP was observed by fluorescence microscopy and the relative fluorescence volume was measured by multifunctional microplate reader.Result: 1.Systematically analyzed the Shenzhen district and global H5 /H7 / H9 and N2 / N6 / N9-UTR sequence characteristics and induced mutated UTR sequence features 2.The constructed RNA polymerase I system has been verified to efficiently drive the expression of the EGFP reporter gene as a functional study of the RNA polymerase I fluorescence reporter system for UTR mutations.3.Successfully constructed the H5/H7/H9 and N2/N6/ N9-UTRrandom mutagenesis gene libraries and the mutated capacity was achieved the construction requirements.4.The experimental group H7-10 / H9-1 / N2-5 UTR mutation can significantly up-regulate the expression of the reporter gene.Conclusion: The RNA polymerase I fluorescence reporting system,which does not contain any RNA promoter,was successfully used to study the function of influenza virus HA/ NA-UTR.The random mutagenesis gene libraries of HA / NA-UTR were successfully constructed with the capacity of 106 cfu / m L.Randomly selected positive clones H7-10 / H9-1 / N2-5 UTR mutations can significantly up-regulate the expression of the reporter gene.In order to further explore the impact of UTR polymorphism sites on the expression of HA and NA genes in influenza A virus,it is suggested that the packaging of HA / NA highly expressed influenza A virus can lay the foundation for the development of influenza.