Recombinant mouse beta-alexin 3 polypeptide, preparation and use thereof
A defensin, recombinant plasmid technology, applied in biochemical equipment and methods, recombinant DNA technology, microorganism-based methods, etc., to achieve high differential virulence, reduce production costs, and facilitate industrial production.
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Embodiment 1
[0029] Embodiment 1: Preparation of recombinant mouse β-defensin 3 polypeptide
[0030] 1. Obtain the mouse Mbd-3 gene
[0031] (1) Use Trizol reagent (purchased from Invitroge, USA) to extract the total RNA of the respiratory epithelium of mice injected with Escherichia coli LPS (purchased from Sigma, USA) for 12 hours;
[0032] 1) BALB / c mice were intraperitoneally injected with LPS at a dose of 2.5 mg / kg, and the mice were sacrificed by removing eyeballs and bleeding after 12 hours;
[0033] 2) Take 100 mg of mouse lung tissue under sterile conditions;
[0034] 3) Add 1ml Trizol;
[0035] 4) Fully grind in liquid nitrogen, mix by inverting for 10 times, and let stand at room temperature for 5 minutes;
[0036] 5) Add 0.2ml of chloroform, invert and mix for 10 times, and let stand at room temperature for 5 minutes;
[0037] 6) Centrifuge at 12000rpm for 15min at 4°C;
[0038] 7) Take the supernatant (about 0.4ml) and transfer it to another EP tube;
[0039]8) Add an eq...
Embodiment 2
[0090] Example 2: Determination of Anti-filamentous Fungal Activity of Recombinant Mouse β-Defensin 3 Polypeptide
[0091] 1. Preparation of culture medium
[0092] (1) 1 / 2 potato dextrose medium (1 / 2PDB: 10% potato, 1% glucose, natural pH), to which tetracycline (final concentration 10 μg / ml) and cefotaxime (final concentration 100 μg / ml) were added.
[0093] (2) Sabouraud dextrose agar medium (1% peptone, 2% glucose, natural pH)
[0094] 2. Preparation of filamentous fungal strains
[0095] Inoculate Aspergillus flavus (clinical isolate), Aspergillus niger (clinical isolate), Microsporum canis (clinical isolate), and Trichophyton rubrum (clinical isolate) on the Sabouraud dextrose agar medium, Grow at 28°C for 7-10 days.
[0096] 3. Preparation of experimental bacteria
[0097] Take about 5 mg each of Aspergillus flavus, Aspergillus niger, Microsporum canis and Trichophyton rubrum, place them in 5ml 1 / 2PDB medium, blow and blow 10 times with a sterile pipette, and take t...
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