Amylin compatible polypeptide and application thereof
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A technology for affinity and peptides, applied in the field of Amylin affinity peptides and its applications, can solve the problems of lack of affinity inhibitors
Active Publication Date: 2019-09-17
EAST CHINA UNIV OF SCI & TECH
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Although there are many types of Amylin inhibitors, there is stil
Method used
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Embodiment 1
[0024] The screening of the affinity polypeptide of embodiment 1.Amylin
[0025] 1. Screening of affinity peptides for Amylin
[0026] ① Preparation of target and activation of host bacteria:
[0027] a. Streak inoculate the strain ER2738 preserved in glycerol on the LB-Tet plate, and culture it upside down at 37°C overnight. Store in the dark at 4°C after sealing with parafilm.
[0028] b. Prepare 6 small sterilized polystyrene Petri dishes, add 1.5mL of 100μg / mL streptavidin solution (dissolved in 0.1M NaHCO pH 8.6 3 ), and rotate well to wet the surface.
[0029] c. Incubate overnight at 4°C with slight shaking in a humidified container, and store at 4°C for later use.
[0030] d. Pick ER2738 monoclonal clone from the LB-Tet plate and inoculate it into 20 mL LB liquid medium, and culture it in a 250 mL Erlenmeyer flask at 37°C with vigorous shaking to the pre-logarithmic stage.
[0031] e. Take a well-coated board, pour out the coating solution, and shake the board vig...
Embodiment 2
[0096] Example 2.LA12 has an inhibitory effect on the process of Amylin aggregation to form fibers
[0097] Molecular simulation predicts that the binding site between Amylin and LA12 contains the hotspot sequence where Amylin aggregates to form a β-sheet, and isothermal titration calorimetry experiments show that Amylin has a strong affinity with LA12. Based on this, LA12 can be used in the research of Amylin inhibitors .
[0098] ThT fluorescence detection:
[0099] Amylin treatment: Dissolve the lyophilized powder in hexafluoroisopropanol to prepare a 1mg / mL peptide stock solution, sonicate in a water bath for 5min, dry it in vacuum, and redissolve it in 10mM Tris-HCl buffer at pH 7.3 , diluted to 30μM (final concentration), dispensed into each EP tube, and shaken at 220rpm / min at 37°C for about 24h. The sampling time points were 0h, 2h, 8h, 12h, 18h, and 24h. Figure 8 . The analysis shows that the entire growth process of Amylin can be clearly divided into two stages: ...
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Abstract
The invention provides Amylin compatible polypeptide and an application thereof. The amino acid sequence of the compatible polypeptide is LTPHKHHKHLHA. Firstly the compatible polypeptide sequence of specific binding Amylin is obtained through a phage display library and a biopanning technique, then Amylin and compatible polypeptide are subjected to molecular simulation and constant temperature titer thermal analysis, which indicates that the interaction site of the compatible polypeptide and the Amylin comprises that Amylin aggregation forms hot spot sequence of beta-sheet, and the affinity and the combination specificity of the Amylin and the compatible polypeptide are high. The AFM and ThT fluorescence detection experiment indicates that the compatible polypeptide has obvious restraining effects on the process of forming fibers through Amylin aggregation, and certain theoretical basis and test basis are provided for development of Amylin aggregation inhibitors and drugs relevant with non-insulin dependent diabetes.
Description
technical field [0001] The present invention relates to the fields of biomedicine and biomaterials, more specifically, relates to an Amylin affinity polypeptide and its application. Background technique [0002] Amylin, also known as human amylin, is a polypeptide composed of 37 amino acids, its sequence is KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 5), its molecular weight is 3906.33g / mol, and it has a positive charge as a whole. Its C-terminus has an amido group, and the cysteines at the second and seventh positions form an intramolecular disulfide bond. These two features are of great significance to the integrity of Amylin's biological functions. However, studies have shown that partially misfolded Amylin itself has a tendency to aggregate to form amyloid fibrils, and these aggregated amyloid fibrils will be deposited in pancreatic beta cells. [0003] Phage display technology is to fuse the foreign DNA fragment encoding the target protein or polypeptide with the...
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