| Influenza A virus is single-stranded, negative sense RNA viruses causing human and animal influenza, which can not only pose a threat to human or animal health, but also result in seriously losses of economic to society. There are two effective measures can be taken to prevent and control influenza A virus. First, influenza vaccination, the effect of influenza vaccine maybe ineffective because there are various subtypes of influenza and Influenza A virus antigen often drift. The other one is the development of antiviral drugs. However, the antiviral of these drugs will decreased rapidly because of drug resistance. Therefore, it’s urgent to develop new methods to prevent and control the influenza A virus. With the important role of host factors in the process of viral replication have been reported, people pay more and more attention to the relationship between host proteins and influenza replication. Phosphorylation of is a means of modification process of protein after the post-translated. Phosphorylation occurs not only within eukaryotic cells protein, but also in the influenza virus proteins and play an important role. Thus, we focus on the host factors impacting on the overall level of phosphorylation of a host cell, such as phosphatase or kinase, hoping to find new host proteins which can resist to drug targets of influenza virus.CDC25B (the cell division cycle 25B) is an important member of CDC25 kinase family, encoding tyrosine phosphatase and plays a crucial role in cell cycle through phosphorylation and phosphorylation, but it could results in tumorgenesis while be overexpressed. Moreover, CDC25B can involve in the process of virus replication. In order to understand the regulatory effect of CDC25B on the replication of influenza A virus (IAV), we used CRISPR/Cas9 system to construct a HELA cell line with CDC25B low expression (CDC25B-/-HELA).When CDC25B-/-HELA cell or WT HELA cell were infected by A/WSN/33(WSN),we found that the viral replication and transcription were downregulated significantly, NP protein were detained in nucleus, and vRNP polymerase activity decreased significantly. The results suggest that CDC25B support the replication and transcription of influenza A virus (IAV) infection in which further illustrate the regulatory mechanism of host protein of influenza virus, and provide possible targets for antiviral drugs research.In addition to vaccineand antiviral drugs injection, detecting and diagnosing virus early is a also very important method to control Influenza A effectively. It provide the prerequisite for the treatment of type A influenza virus. Therefore, A influenza virus NA protein was used as an antigen in this study to establish an indirect ELIS A method for the rapid detection of influenza A virus subtypes. Homology was compared according to the relevant nucleotide sequence of H5N1 Subtype influenza virus found from the GenBank. The target combinant protein is the NA amino acids (139-400) of virus A/duck/Shandong/009/2008 (H5N1) strain was expressed and purified in the E. coli BL21 (DE3).The purified recombinant protein as antigen to construct indirsect-ELISA of NA Protein of influenza A virus. The optional working circumstances for the ELISA assay,N1 antigen concentration 400 ng/well, Serum dilution 1:20, the second antibody dilution 1:5000.The positive serum OD450=15.579>2.1,it shows that the recombinant protein has a strong antigenicity. Regarding sensitivity, specificity, reproducibility and correlation with other methods, the developed indirect ELISA, it will provide some scientific basis for detection and diagnosis of influenza virus subtype Nl, and be contribution in resistance to influenza virus infection. |
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