A mutant of pig natural immune protein lgp2 and its preparation and use
A technology of natural immunity and mutants, which is applied in the fields of peptide/protein components, animal/human proteins, medical preparations containing active ingredients, etc. It can solve problems such as unclear performance, infection, and low infection rate of wild-type mice
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Embodiment 1
[0028] Example 1 Construction of antiviral plasmid
[0029] (1) Extraction of total RNA from porcine cells
[0030] Pig kidney epithelial cells (PK-15) were cultured in a 35mm cell culture dish. After the cells were congested, the supernatant was discarded, and the cells were washed 3 times with PBS.
[0031] Add 1 mL TRIzol to lyse (suspend cells by pipetting), and let stand at room temperature for 5 minutes.
[0032] Add 250 μL of chloroform, shake vigorously, place at 4°C for 10 min, and extract RNA.
[0033] Centrifuge at 12,000 rpm at 4°C for 15 minutes; pipette 450 μL of supernatant, transfer to a new centrifuge tube, and add an equal volume of isopropanol. Precipitate for 20 minutes at -20°C.
[0034] Centrifuge at 12,000 rpm at 4°C for 10 minutes; discard the supernatant, add 1 mL of 75% ethanol, and shake gently by inversion 3 times. Wash away impurities such as salt particles.
[0035] Centrifuge at 10,000 rpm at 4°C for 5 minutes; discard the supernatant, dry a...
Embodiment 2
[0089] Example 2 Screening and application of antiviral proteins
[0090] (1) Amplification of foot-and-mouth disease virus
[0091]Spread BHK cells (hamster kidney cells) into T25 cells. After forming a single layer of cells, discard the cell culture medium, wash 3 times with PBS, then inoculate 1MOI foot-and-mouth disease virus, absorb for 1 hour, and shake once every 20 minutes. To ensure uniform adsorption of virus. After the adsorption, the virus supernatant was discarded, washed once with PBS, the unadsorbed virus was absorbed, and 2.5 mL of cell culture maintenance solution (MEM medium containing 0.5% serum) was added. Observe the lesions, collect the cells and supernatant after the cells are completely detached, freeze and thaw three times, collect the virus liquid by centrifugation, and freeze at -80°C for later use. O / BY / CHA / 2010 and Asia1 / HN / 2006 strains were respectively amplified by the above amplification method.
[0092] (2) Research on the inhibition of foot...
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