Polypeptides and their genes and applications

A technology of printing mouse and flea, applied in the field of biomedicine, can solve the problem that there are not many studies on pharmacological active components, and achieve the effect of inhibiting migration

Active Publication Date: 2020-07-17
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] my country has a long history of medicinal research on the toxins of scorpions, snakes, spiders, toads, centipedes, etc., but there are not many studies on pharmacologically active components in blood-sucking arthropods such as fleas

Method used

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  • Polypeptides and their genes and applications
  • Polypeptides and their genes and applications
  • Polypeptides and their genes and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of the Polypeptide FS48 Gene of the Passenger Flea

[0028] 1. Extraction of total RNA from the salivary glands of the imprinted fleas: live imprinted fleas were placed on ice for 30-60 minutes for freezing and anesthesia, and 50 imprinted fleas were dissected under a microscope to obtain the salivary glands, and 1 mL of total RNA extraction buffer (Trizol solution, Trizol solution, U.S. GIBCOBRL company product), homogenize 1 minute in 5m1 glass homogenizer. Add an equal volume of phenol / chloroform solution, mix vigorously, place at room temperature for 1 minute, centrifuge at 12,000 rpm for 10 minutes at 4°C, and discard the precipitate. Add an equal volume of isopropanol to the supernatant, let it stand at room temperature for 4 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it in the air.

[0029] II. Purification of mRNA from the salivary glands of the imprinted mouse flea: the isolatio...

Embodiment 2

[0034] Escherichia coli expression of embodiment 2 Indian mouse flea polypeptide FS48

[0035] Chemically transform the expression vector plasmid containing the gene of P. indica polypeptide FS48 into BL 21 (DE 3 ) pLysS recombinantly expressed in Escherichia coli, insert 10ml of overnight culture seed bacteria solution into 1L LB medium, cultivate at 37°C for 3 hours, when the light absorbance value of the bacteria is OD 600 When it reaches about 0.8, add IPTG to a final concentration of 1mM, continue to cultivate for 3 hours, collect the bacteria by centrifugation at 8000rpm for 10 minutes, suspend the sediment with 100ml of 25mM Tris-HCl solution with pH 8.0, then centrifuge at 8000rpm for 10 minutes to collect the bacteria, and remove the supernatant , add 40ml of 25mM Tris-HCl solution with pH 8.0 to the pellet, ultrasonically break the cells at 4°C, super 30s and stop for 30s 4 times in a row, then centrifuge at 12000rpm for 30 minutes to collect the supernatant, dialyz...

Embodiment 3

[0036] Example 3 Isolation and Purification of the Expression Product of Escherichia coli FS48 Escherichia coli

[0037] The first step is Hiprep SP cation exchange chromatography column chromatography. According to the above method, the Escherichia coli expression product of Pa. musculus polypeptide FS48 was dialyzed and then centrifuged at 14000rpm at 4°C for 60min to remove the precipitate. The supernatant was loaded on the Hiprep SP cation-exchange chromatographic column of GE Company. Hiprep SP cation exchange column with 20mM Na 2 HPO 4 -NaH 2 PO 4 Buffer equilibration at pH 6.0, obtained by elution with a linear gradient from 0% to 100% in the same buffer containing 1M NaCl figure 2 A, Collect active peaks as shown.

[0038] The second step is Sephadex SR-100 gel filtration column chromatography. The active peak obtained by Hiprep SP cation exchange chromatography was concentrated by a 3kDa ultrafiltration tube and loaded on a Sephadex SR-100 gel filtration colu...

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Abstract

The invention relates to Xenopsylla cheopis polypeptide and a gene and application thereof, in particular to application of Xenopsylla cheopis polypeptide FS48 in preparation of medicine for treating pain, cancer, inflammatory diseases, neurodegenerative diseases, cardiovascular diseases, autoimmune diseases and chronic infectious diseases related to Kv1.1. The amino acid sequence of the Xenopsylla cheopis polypeptide FS48 is shown as SEQ ID NO:1. The polypeptide is prepared by conducting prokaryotic expression of Xenopsylla cheopis polypeptide FS48 and then conducting three-step column chromatography separation and purification. The Xenopsylla cheopis polypeptide FS48 has the pain relief, anti-inflammation, immunoregulation and anti-tumor effects by inhibiting Kv1.1 and Kv4.1 and has no cytotoxicity.

Description

Technical field: [0001] The invention relates to the field of biomedicine, in particular to a protein obtained from animal tissue and its application. Background technique: [0002] Potassium channel is a type of transmembrane protein widely distributed in cells and tissues such as skeletal muscle, nerve, heart, blood vessel, gland, etc. It plays an important role in the signal transmission process of many excitatory and non-excitatory cells, such as , action potential regulation, cardiac pacing, signal integration, hormone release, cell volume regulation, and cell proliferation (Curr Opin SupportPalliat Care, 2015Jun; 9(2):147-54). In addition, the role of potassium channels in cell proliferation and growth in prostate, colon, lung and breast cancers was also discovered (Annu Rev Cell Dev Biol, 2015; 31:231-47). At present, about 80 different potassium channel subtypes have been reported, among which Kv1.1 is in CD 4+ It is expressed on helper T cells, vascular smooth mus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70A61K38/17A61P35/00A61P29/00A61P25/28A61P9/10A61P31/00
CPCA61K38/00C07K14/43504C12N15/70C12N2800/101
Inventor 徐学清张贝邓振晖
Owner SOUTHERN MEDICAL UNIVERSITY
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