Amphibian-animal-source bradykinin and application thereof
A technology of bradykinin and Hainan turbulent frog, applied in the field of biomedicine, to achieve the effect of promoting ileal muscle contraction and strengthening ileal muscle contraction
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Embodiment 1
[0023] Cloning of bradykinin-AH gene from Hainan turbulent frog:
[0024] 1) Extraction of total RNA from the skin of the Hainan turbulent frog:
[0025] ①Take 300mg of Hainan Turbulent frog skin tissue, put it into a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1m1 of total RNA extraction buffer (Trizol, product of Invitrogen, USA), mix well, and then in 4 Centrifuge at 12000rpm for 10min.
[0026] ② Centrifuge to take the supernatant, add 0.2ml chloroform solution, mix vigorously, let stand at room temperature for 10 minutes, then centrifuge at 4°C, 12000rpm for 10 minutes, and discard the precipitate.
[0027] ③ Add an equal volume of isopropanol to the supernatant, let it stand at room temperature for 10 minutes, centrifuge at 4°C and 12,000 rpm for 10 minutes, collect the precipitate, wash it once with 75% (V / V) ethanol, and dry it. The precipitate at the bottom of the tube is Hainan Turbulence Total RNA from frog skin.
[0028...
Embodiment 2
[0055] Chemical Synthesis of Bradykinin-AH from Hainan Frog Rana:
[0056] Ⅰ. The chemical synthesis method of bradykinin-AH from Hainan turbulent frog: According to the mature peptide amino acid sequence deduced from the gene, its complete sequence was synthesized with an automatic peptide synthesizer (433A, Applied Biosystems), and desalted by HPLC reverse-phase column chromatography.
[0057] Ⅱ. Molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF).
[0058] Ⅲ. The purity of the purified bradykinin-AH of Hainan turbulent frog was identified by high performance liquid chromatography (HPLC), the molecular weight was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), and the isoelectric point was determined by isoelectric focusing electrophoresis. The amino acid sequence structure was determined by an automatic amino acid sequencer.
[0059] Bradykinin-AH...
Embodiment 3
[0061] Bradykinin-AH Activity Experiment of Hainan Turbulent Frog Bradykinin:
[0062] 1. Detection of the activity of Bradykinin-AH in promoting ileal muscle contraction:
[0063] Take a guinea pig (body weight 150-250g, sex random) about 10cm long terminal ileum muscle, put it into the table-top solution quickly (the table-top solution is 137mM NaCl, 2.7mM KCl, 1.36mM CaCl 2 ,0.49mM MgCl 2 ,0.36mM NaH 2 PO 4 ,11.9mM NaHCO 3 , 5.04mM D-glucose). Cut the isolated guinea-pig ileum muscle into 2cm fragments in a bench-top solution, ventilate it with air, load a weight of 1g, keep warm at 37°C, and after the muscle contraction and relaxation are stable, add final concentrations of 0, 2, 4, 8, 16 , 32ng / ml of the bradykinin-AH sample was measured by a signal processing system in the contraction state of the isolated guinea pig ileum muscle (see figure 1 ). The signal collection and processing adopts the PcLab biological signal processing system produced by Beijing WeChat St...
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