A Short Peptide Inhibitor Inhibiting the Aggregation of Amylin
A technology of amyloid peptide and inhibitor, which is applied in the field of biological preparations, can solve the problems of rare species, low inhibition rate, and large molecular weight, and achieve the effects of convenient drug use, reduced toxicity, and small molecular weight
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Embodiment 1
[0030] Example 1 Effects of Different Concentrations of Inhibitors on the Aggregation of IAPP Cultured in Vitro
[0031] (1) Reagent preparation
[0032] Take out the IAPP, ANFLVH and the short peptide inhibitor FLPNF of the present invention stored in the refrigerator at -80°C and place them at room temperature. Among them, ANFLVH and the short peptide inhibitor FLPNF of the present invention are synthesized by entrusting a biological company, lyophilized at 1 mg / tube pink.
[0033] A ThS solution with a concentration of 0.5wt.% was prepared with deionized water for later use.
[0034] IAPP was dissolved in the above-mentioned 0.5 wt.% ThS solution to make the IAPP concentration 40umol / L, and the IAPP solution was prepared.
[0035] Dissolve ANFLVH in the above ThS solution, prepare ANFLVH concentrations of 300umol / L, 400umol / L and 500umol / L, respectively, and prepare ANFLVH solutions.
[0036] FLPNF was dissolved in the above ThS solution so that the concentrations of FLP...
Embodiment 2
[0039] Example 2 Effects of Inhibitors on the Aggregation of IAPP in INS1 (Rat Insulinoma) Cells (hIAPP-INS1) Transfected with Human IAPP
[0040] Take a 96-well plate and dilute INS1 (rat insulinoma cells) with culture medium to a density of 5×10 according to the conventional cell culture method. 4 cells / mL, cells were plated, and a blank control group, a positive control group, and an experimental group were set up in three groups, and 30 wells were selected in the middle area, with 10 wells in each group and 100 μL in each well, that is, 5000 cells / well, and placed in a 37°C, 5%CO 2 After the cell incubator was adhered to the wall, the medium was changed, and the human IAPP gene transfection test was carried out according to the conventional cell transfection method to form INS1 cells transfected with human IAPP, namely the test cell hIAPP-INS1.
[0041] After successful transfection, the blank control group was added with 100 μL of culture fluid, the positive control grou...
Embodiment 3
[0043] Example 3 Effects of Different Concentrations of Inhibitors on the Aggregation of IAPP in INS1 (Rat Insulinoma) Cells (hIAPP-INS1) Transfected with Human IAPP
[0044] Take the 96-well plate, according to the conventional cell culture method, the rat insulinoma cell INS1 was dispersed and diluted with the culture medium to a western density of 5×10 4 cells / mL, add 5,000 cells to each well of the 60 wells in the middle, and put them in 37°C, 5% CO 2 After the cell incubator was adhered to the wall, the medium was changed and the human IAPP gene transfection test was carried out according to the conventional cell transfection method to obtain cells for the hIAPP-INS1 test.
[0045] After successful transfection, co-culture experiments with different concentrations of short peptide inhibitors and cells were carried out, and 5 experimental groups with different concentration gradients were set up. At the same time, a blank control group cultured in ordinary culture medium ...
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