Lychas mucronatus Kv1.3 blocker gene, preparation method and application
A technology of kv1.3 and blocking agent, applied to recombinant Escherichia coli and its construction and peripheral injury medicine of Scorpion scorpion Kv1.3 blocker The Kv1.3 blocker gene of the scorpion scorpion, the preparation of the Kv1.3 blocker of the scorpion, the isolation of the Kv1.3 blocker gene of the scorpion, and the role of the Kv1.3 blocker in autoimmune diseases Field of use
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Embodiment 1
[0040] Example 1: Extraction of total RNA from scorpion venom glands (Trizol LS one-step method: Trizol LS is purchased from Invitrogen in the United States) ① Take 500 mg of scorpion glands and grind them into fine powder in liquid nitrogen, add 10ml TRIZOL reagent and mix well, room temperature (20-25 ℃, the same below) and let stand for 5 minutes; ② Then add 2ml chloroform and mix for 15 seconds, let stand at room temperature for 2-3 minutes, and centrifuge at 12000g for 15 minutes at 4°C; Centrifuge at 12000g for 10 minutes at ℃ to obtain RNA precipitate; ④ wash the precipitate with 5ml of 75% ethanol, centrifuge at 7500g for 5 minutes; ⑤ dissolve the RNA precipitate in DEPC-treated water after drying, and incubate at 55-60℃ for 10 minutes to completely dissolve the RNA. The whole process was carried out according to the method recommended by TRlZOL (Total RNA Isolation) Reagent Kit.
Embodiment 2
[0041] Embodiment 2: Separation and purification of mRNA
[0042] The PolyA Tract mRNA isolation system (Promega, USA) was used to isolate and purify mRNA. Its working principle is based on the complementary pairing characteristics of Oligo(dT) and the poly(A) tail at the 3' end of mRNA. Oligo(dT) was labeled with biotin and passed It anneals with the mRNA 3' end poly(A) to form a hybrid, then captures and washes the biotin Oligo(dT) / mRNA hybrid with avidin-labeled magnetic beads and a magnetic separation rack, and finally uses RNase-free wxya 2 O elutes it to achieve the purpose of isolating mRNA from total RNA. ① Sample preparation: RNA was added to 800 μl GTC containing 32 μl β-mercaptoethanol. ②Annealing of the probe: Take 5 μl of Oligo (dT) at a concentration of 250 pM, add distilled water to 50 μl; add 1.6 ml of preheated (70°C) dilution buffer (32 μl β-mercaptoethanol has been added to the dilution buffer), and mix with RNA, 70 Incubate for 5 minutes at °C. ③Activat...
Embodiment 3
[0043] Example 3: First strand cDNA synthesis
[0044]①Add 2μl Not I Primer-adapter (INVITOGEN, USA) and 6μl mRNA (including 3μg mRNA) to a 1.5ml Ep tube, incubate at 70°C for 10min, put it on ice quickly, and after centrifugation, add the following components: 4μl 5X first strand buffer (INVITOGEN, USA); 2μl 0.1MDTT; 1μl 10mM dNTPs; 1μl DEPC·H 2 O. Gently mix and centrifuge, put at 37°C for 2 min; ② Add 5 μl reverse transcriptase, take 2 μl after mixing, add 1 μl [α- 32 P] dCTP (4 μCi) (tracer tube). Incubate with the above reaction components (sample tubes) at 37°C for 1 hour, and then put them on ice to terminate the reaction; ③ For the tracer tubes, add 43 μl 20 mM EDTA and 5 μl yeast tRNA in sequence, mix well, and take two 10 μl portions respectively. On two filter membranes, wash 1 part with 10% TCA 3 times, each time for 5 minutes, wash 1 time with 95% ethanol, air dry, put into 1.5ml scintillation fluid (1 # sample); the other 1 part was air-dried and put into 1.5...
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