uba52 protein and its application
A gene and inhibitor technology is applied in the application field of UBA52 protein in regulating the replication of H5N1 avian influenza virus, which can solve the problems to be further studied and achieve the effect of inhibiting replication efficiency.
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Embodiment 1
[0020] Example 1: Verification of the interaction relationship between host protein UBA52 and H5N1 avian influenza virus proteins PA, PA-N155 and PA-N182
[0021] 1. Construction of PA, PA-N155, PA-N182 and UBA52 gene expression vectors
[0022] According to the pcDNA3.1-3×Flag C carrier information and the PA, PA-N155 and PA-N182 gene sequences of A / Chicken / ShanXi / 2 / 2006 (H5N1) published in the Influenza research database, primers were designed using Oligo6 software, and the gene Both upstream and downstream primers contain restriction enzyme sites NheI and BamhI sequences.
[0023] According to the pcDNA3.1-Myc vector information and the chicken UBA52 gene (GenBank accession number: NM_205075.1) 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 ...
Embodiment 2
[0053] Example 2: siRNA interferes with the expression of host protein UBA52
[0054] 1. siRNA design and experimental grouping
[0055] Two siRNAs were designed for the UBA52 gene. The sequences siUBA52-1, siUBA52-2, and siNC were synthesized by Guangzhou Ruibo Biological Company. The siRNA sequences are shown in the table below.
[0056]
[0057] The experiment was divided into three groups, experimental group (siUBA52), control group (siNC), no treatment group (MOCK). The transfection amount of siUBA52 was 100 nM, that is, two siUBA52s were 50 nM each. siRNA was transfected into DF1 cells by lipofection 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...
Embodiment 3
[0062] Example 3: UBA52 regulates H5N1 avian influenza virus replication
[0063] 1. Virus titer detection (TCID50assay)
[0064] The replication efficiency of the progeny virus of H5N1 avian influenza virus was evaluated by TCID50 assay, and the experiment was repeated three times.
[0065] (1) After siUBA52 was transfected into DF1 cells for 24 hours, the cells were washed twice with serum-free medium;
[0066] (2) The virus is diluted with a maintenance medium containing 1% fetal bovine serum, and the cells are infected with 0.01MOI inoculated dose;
[0067] (3) After virus adsorption for 1 hour, wash 2 times with serum-free medium, add 2 ml of maintenance medium, and collect supernatant at 12 hours and 24 hours;
[0068] (4) In a 1.5mL EP tube, the virus supernatant obtained in step (3) was serially diluted 9 times with serum-free medium, that is, 10 -1 ~10 -9 ; Discard the cell culture medium in the 96-well plate at the same time, wash 2 times with 1x PBS, add virus s...
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