Application of stau2 in regulation of avian influenza virus replication
A bird flu virus and bird flu technology, applied in the field of genetic engineering, can solve problems that need further research and achieve the effect of inhibiting replication efficiency
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Embodiment 1
[0021] Example 1: Verification of the interaction relationship between host protein STAU2 and H5N1 avian influenza virus protein NS1
[0022] 1. Construction of NS1 and STAU2 gene expression vectors
[0023] According to the pcDNA3.1-3×Flag C carrier information and the NS1 gene sequence of A / Chicken / ShanXi / 2 / 2006 (H5N1) published in the Influenza research database, the primers were designed using Oligo6 software, and the upstream and downstream primers of the gene contained restriction Restriction sites NheI and EcoRI sequences.
[0024] According to the pcDNA3.1-Myc vector information and the chicken STAU2 gene (GenBank accession number: NM_001030941.1) sequence published on NCBI, the primers were designed using Oligo6 software, and the upstream and downstream primers also contained NheI and BamhI restriction sites, respectively. The sequences of the above primers are shown in the table below, and the underlined part indicates the enzyme cleavage site.
[0025]
[0026]...
Embodiment 2
[0054] Example 2: siRNA interferes with expression of host protein STAU2
[0055] 1. siRNA design and experimental grouping
[0056] siRNA was designed for the STAU2 gene, and the sequences siSTAU2-1, siSTAU2-2, and siNC were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. The siRNA sequences are shown in the table below.
[0057]
[0058] The experiment was divided into three groups, experimental group (siSTAU2), control group (siNC), no treatment group (MOCK). The transfection amount of siSTAU2 was 100 nM. siRNA was transfected into DF1 cells by liposome transfection method, and transfected when the cells were cultured to 80% confluence. cell transfection press 3000 reagent (Life Technologies) instructions. use Medium dilution 3000 reagent and mix well, use Dilute the siRNA in the culture medium and mix well, put the diluted siRNA in each tube The 3000 reagent was mixed with the siRNA premix, incubated at room temperature for 10 minutes, added to the c...
Embodiment 3
[0063] Example 3: STAU2 regulates replication of H5N1 avian influenza virus
[0064] 1. Virus titer detection (TCID50assay)
[0065] The replication efficiency of the progeny virus of H5N1 avian influenza virus was evaluated by TCID50 assay, and the experiment was repeated three times.
[0066] (1) 24 hours after transfection of siSTAU2 into DF1 cells, wash 2 times with serum-free medium;
[0067] (2) The virus is diluted with a maintenance medium containing 1% fetal bovine serum, and the cells are infected with 0.01MOI inoculated dose;
[0068] (3) After virus adsorption for 1 hour, wash 2 times with serum-free medium, add 2 ml of maintenance medium, and collect supernatant at 12h and 24h;
[0069] (4) In a 1.5mLEP tube, use serum-free medium to make serial 9-fold dilutions of the virus supernatant obtained in step (3), that is, 10-1 to 10-9; at the same time, discard the 96-well plate in which MDCK cells were cultured. After washing 2 times with 1xPBS, add virus suspensio...
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