Recombinant lamprey lysin as well as preparation method and application thereof in preparing antithrombotic medicament
A technology of lamprey and lysin, which is applied in the field of medicine and medical engineering, to achieve enhanced antithrombotic effect, large safety range, and mild toxicity
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[0061] a. Preparation of eellysin gene:
[0062] (1) The oral glands of Japanese lamprey were taken and quickly stored in liquid nitrogen;
[0063] (2) Weigh 0.2g lamprey oral gland, add 1ml TRIzol reagent to prepare venom gland homogenate, incubate at 4°C for 5min;
[0064] (3) Add 0.2ml chloroform, close the cap tightly, shake for 15sec, and place on ice for 5min;
[0065] (4) 4°C, 12000xg, centrifuge for 15 minutes;
[0066] (5) Transfer the upper aqueous phase to another centrifuge tube, add 0.5ml of isopropanol, and incubate on ice for 10 minutes;
[0067] (6) 4°C, 12000xg, centrifuge for 15 minutes;
[0068] (7) Discard the supernatant, add 1ml 75% ethanol to the precipitate to wash, and vortex to mix;
[0069] (8) Centrifuge at 10,000xg for 5 minutes at 4°C to obtain the RNA precipitate of eellysin;
[0070] (9) After air drying, dissolve RNA with TE or RNase-free water for later use;
[0071] b. Perform RT-PCR amplification to obtain the target cDNA;
[0072] Th...
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