Application of interferon kappa in preparation of anti-enveloped virus medicines
An interferon and envelope technology, applied in the field of biomedicine, can solve the problems of public health safety hazards, inability to determine IFN-κ enveloped virus, inconsistent replication mechanism, etc.
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Embodiment 1
[0014] Example 1: Construction of pSV1.0-IFN-κ overexpression plasmid
[0015] The present invention has cloned IFN-κ from the human genome, the nucleotide sequence of its coding gene is shown in SEQ ID No.1, the full-length amino acid sequence is shown in SEQ ID No.2, and the IFN-κ described in the present invention Belonging to the type I interferon family, it has only 30% homology with IFN-α and IFN-β.
[0016] In order to study the function of IFN-κ, we constructed the eukaryotic expression vector of IFN-κ, and eukaryotically expressed the mature secreted protein in cell lines in vitro, and then detected the effect of IFN-κ protein on the replication of influenza virus and Zika virus influences. First, we used the eukaryotic expression vector pSV1.0 to construct the IFN-κ eukaryotic expression plasmid. The construction method was as follows: the cDNA generated by reverse transcription of RNA extracted from A549 cells was used as a template, and the corresponding primers w...
Embodiment 2
[0020] Example 2: IFN-κ inhibits the replication of influenza viruses H7N9, PR8 and H9N2
[0021] The lung epithelial cell line A549 is derived from human non-small cell lung cancer epithelial cells, which is the main cell model for studying influenza virus infection. In order to verify the effect of IFN-κ on different subtypes of influenza virus infection, this example uses 12-well plates The pSV1.0-IFN-κ overexpression plasmid and the control plasmid pSV1.0-GFP were transfected in the A549 cell line. After 24 hours, 100 μL of three influenza virus PR8 ( figure 2 a), H9N2 ( figure 2 b), and H7N9 ( figure 2 c), the average number of virus-infected particles per cell (MOI) is 1. in CO 2 After continuing to incubate in the incubator for 2 hours, discard the virus solution, wash it twice with PBS, add DMEM complete medium and continue to culture for 48 hours, then collect the cells, and analyze the expression of IFN-κ and the expression of influenza virus nucleoprotein NP a...
Embodiment 3
[0023] Example 3: IFN-κ inhibits the replication of Zika virus
[0024] Astrocyte U-251 cells are one of the main target cells of Zika virus infection. In order to verify the effect of IFN-κ on Zika virus infection, U-251 cells were used to transfect control plasmids pSV1.0 and pSV1.0-IFN-κ plasmids. After 36 hours of transfection, cells in some wells were harvested to detect IFN in the cells -κ protein expression level ( image 3 a-left). The remaining cells were infected with Zika virus at a multiplicity of infection (MOI = 2), infected at 37 °C for 2 h, washed twice with PBS, and replaced with fresh DMEM medium containing 2% FBS to continue culturing. After 36 hours of Zika virus infection, the cells were harvested, and a part of the cells were used for western blot (WB) analysis of the expression levels of Zika virus non-structural proteins (NS2b, NS3, NS5) in U-251 cells ( image 3 b-left). Another part of cells was analyzed by immunofluorescence staining technique to...
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