Micro-molecule polypeptide as well as coding gene and application thereof
A technology of small molecule polypeptides and genes, applied in small molecule polypeptides and their coding genes, and in the preparation of drugs for the treatment of chronic inflammatory diseases, can solve problems such as loss, intestinal epithelial cell apoptosis, and impaired ability to inhibit apoptosis
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Embodiment 1
[0033] Example 1: Preparation of Small Molecular Peptides
[0034] The amino acid sequence of the small molecule polypeptide is shown in SEQ ID NO.1. The small molecule polypeptide was synthesized on an ABI 431A solid-phase peptide synthesizer (PE Company, USA). Methods The standard fluorenylmethoxycarbonyl (Fmoc) protocol was used, and arginine was coupled twice. Initially select 0.125mmol p-hydroxymethylphenoxymethyl polystyrene resin (HMP resin), extend the peptide chain from the carboxyl terminal to the amino terminal one by one according to the polypeptide sequence, the amount of each amino acid is 0.5mmol, and the mole of the resin The ratio is 4:1. The α-amino acids of various amino acids are protected by Fmoc, and the other side chain protection groups are: Ser (tBu), Glu (OtBu), Arg (Pmc). The first amino acid is connected to the resin with 4-lutidine, the amino acid is activated with 1-hydroxybenzotriazole and dicycloethyl carbon, and after coupling, the volume frac...
Embodiment 2
[0038] Example 2 Application of Small Molecular Peptides in the Treatment of Crohn's Disease in Mice
[0039] 1. Animals:
[0040] Female BalB / C mice, weighing 13-14 grams, were purchased from the Animal Laboratory of Nantong University and kept in a clean animal room. 2. Drugs:
[0041] Small molecule polypeptide treatment group: 20nmol / kg body weight of the small molecule polypeptide shown in SEQ ID NO: 1 was given for intraperitoneal injection, a total of 6 mice;
[0042] Blank group: intraperitoneal injection of the same volume of PBS as the treatment group, a total of 6 rats.
[0043] 3. Experimental method:
[0044] Colonic perfusion of TNBS is a common animal modeling method for Crohn's disease. This experiment is used to verify the therapeutic effect of small peptide molecules on Crohn's disease. Colon perfusion TNBS method, specifically:
[0045] 1) Adult healthy 13-14g BALB / C female mice, fasted for 24 hours, intraperitoneally injected with general anesthesia; ...
Embodiment 3
[0067] Example 3 Interaction between SOCS1 and FYN
[0068] 1. Cells: HT-29 cells, purchased from Shanghai Aolu Biological Co., Ltd., the medium used is 1640 complete culture. When used, fetal bovine serum with a final concentration of 10% was added, and 100 units of penicillin and 100 μg of streptomycin were added per milliliter. At 37°C, 5% CO 2 , humidified air cell culture incubator. The medium was changed every two days and passages were performed every 4-5 days. Add EDTA digestion solution (PBS / EDTA: 2g ethylenediaminetetraacetic acid disodium (EDTA-Na2) to 1000ml PBS, adjust the pH value to 7.8 with NaOH until EDTA-Na2 is completely dissolved, and store at 4°C after autoclaving ) to digest the cells until most of the adherent cells fall off, then stop the digestion, and transfer an appropriate amount of cells into a new culture flask after blowing and centrifuging.
[0069] 2. Material method:
[0070] 1)H 2 o 2 Stimulate HT-29 cells, digest with EDTA / PBS buffer ...
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