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@#Objective To construct encoding RNA that can be cyclized in vitro by using the permuted intron exon(PIE)strategy in the maturation process of eukaryotic mRNA,and transfect it into HEK-293T cells for expression.Methods The sequences of 5'and 3'cyclic arms with groupⅠcatalytic intron,the internal ribosome entry sites(IRES)of Coxsackievirus B3(CVB3)and the target gene were selected to construct the template plasmid. Linearization plasmid template obtained by PCR was used to synthesize linear RNA through in vitro transcription(IVT),which then started in vitro cyclization(IVC)by the addition of cyclization reagents to obtain circular RNA(circRNA). RNA cyclization was confirmed by agarose gel electrophoresis and ribonuclease R(RNase R)digestion. HEK-293T cells were transfected with circRNAs respectively carrying enhanced green fluorescent protein(EGFP),firefly luciferase(Fluc),and influenza virus hemagglutinin(HA)IVR-180 genes,to verify their expression with in vitro.Results With RNA cyclization,the main band of agarose gel electrophoresis became smaller and small fragments appeared. After RNase R digestion,only some circRNA bands remained.HEK-293T cells transfected with EGFP-circRNA showed significant green fluorescence under the fluorescence microscope.The Fluc expression values of HEK-293T cells transfected with Fluc-circRNA were on average 20 times higher than non cyclized RNA,and the relative light unit(RLU)scaled up with the increase of Fluc-circRNA transfection dose. Western blot analysis showed that HA protein was successfully expressed in HEK-293T cells transfected with HA-circRNA.Conclusion In this study,linear RNA was successfully cyclized in vitro and different proteins were expressed,which lays a foundation of the research of new influenza vaccines and mRNA vaccines.
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@#At present,the most commonly used method for detecting hemagglutinin(HA)content in influenza vaccines is still single-radial immunodiffusion(SRID). However,the preparation of standards required by this method takes a long time,usually 2 ～ 3 months. Therefore,how to quantitatively analyze HA accurately has always been a difficult problem in the detection of HA content in the situation that reference products can not be obtained at the early stage of the pandemic influenza. High performance liquid chromatography(HPLC)has its own characteristics of rapidity,high sensitivity,good repeatability and high accuracy,which can rapidly determine HA content by using different separation principles and has been widely used in the detection of HA content in influenza vaccine. This paper reviewed the research progress of the application of HPLC in the determination of HA content in influenza vaccine.
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Objective@#To investigate the antigenic epitope prediction method of hemagglutinin (HA) protein of influenza A (H1N1) virus.@*Methods@#BALB/c mice were conventionally immunized with influenza H1N1 vaccine. The splenocytes from the immunized mouse were fused with SP2/0 mouse myeloma cell line, and then the antigen-specific monoclonal antibodies (mAbs) were obtained by screening hybridoma supernatants. ELISA blocking test was used to detect the blocking result of each monoclonal antibody, which was labeled by horseradish peroxidase (HRP). The light and heavy chain variable region genes of each antibody were cloned, the amino acid sites of the antibody-binding HA antigen epitope were predicted by computer simulation.@*Results@#Three hybridoma cell lines of stable secreting anti-H1N1 influenza virus HA protein were obtained.Three mAbs were divided into two categories by ELISA blocking tests, which were divided into two categories according to preliminary results of computer simulation.@*Conclusions@#ELISA blocking test and computer simulation prediction can prove each other in predication of the antigenic epitopes of HA protein of H1N1 influenza virus.
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Objective To investigate the genetic characteristics and mutations in hemagglutinin ( HA) genes of influenza A subtype H3N2 viruses isolated in Fujian province during 2014—2016. Methods HA gene fragments of 44 randomly selected influenza A (H3N2) viruses were amplified by RT-PCR and then sequenced by Sanger sequencing. Obtained sequences were analyzed by bioinformatics software and on-line websites. Results Pair-wise similarity among HA genes of the 44 strains was between 97. 3%-100. 0% at nucleotide level. The average variations between epidemic strains and corresponding vaccine strains in the year of 2014, 2015 and 2016 were 0. 012, 0. 008 and 0. 009, respectively. The genotype of epidemic strains in 2014 was 3C. 3a rather than 3C. 1 of the vaccine strain. Notably, variations at some antigenic sites, re-ceptor binding sites ( RBSs) and N-Glycosylation sites were identified despite the fact that the genotypes were identical between epidemic and vaccine strains in 2015 and 2016. Conclusion Variations at the HA genes of influenza A (H3N2) viruses in Fujian province occurred during the year of 2014—2016, reflecting the ability of circulating strains to escape the vaccine-induced immunity. Sustainable influenza surveillance and prompt identification of viral variants would benefit influenza prevention and control.
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Objective@#Exploring the molecular characteristic of global and Shenzhen district H5N6 and H7N9 influenza viruses HA untranslated regions(UTRs).@*Methods@#Mega7.0 and DNAStar 7.1.0 were used to construct phylogenetic tree and nucleotide analysis.@*Results@#From 2014 to 2015, 3 strains of H5N6 influenza virus from Shenzhen were compared with the other H5NX influenza viruses, the nucleotide homology of HA-3’UTR was 77.4%-100%, which did not have obvious mutated sites. The nucleotide homology of H5N6-HA-5’UTR was 91.7%-100%, and the sites of 24 and 31 sites were mutated. From 2013 to 2014, 11 strains of H7N9 influenza virus from Shenzhen were compared with the other H7NX influenza viruses, the nucleotide homology of H7N9-HA-5’UTR was 76.8%-100%, which had multi-mutated sites on 2-6, 9, 10, 12 and 15-17 positions.@*Conclusions@#HA-UTR from human-infected H5N6 and H7N9 influenza viruses isolated in Shenzhen district has unique molecular characteristics, its conserved region has relatively high homology and the segment-specific region has genetic polymorphism.
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Objective:To search for common epitopes of(H5N1) from different areas in the world in order to develope epitope-bacterin by the way of immunoinformatics.Methods:Firstly,homologous degree of the amino acid sequence of VIA from different areas was compared on the basis of the database in Genebank with soft-ware ClustalW1.83.The common epitopes which were able to bind strongly to MHCⅠ molecule were selected.Results:Though homologous degree of amino acid sequence of VIA from different areas was not completely similar,there were some common epitopes among these strains analyzed.The common epitopes were shown to bind strongly with MHCⅠ molecule.Conclusion:These common epitopes may be used to develope epitope-bacterin for preventing infection of AIV(H5N1).