Extracellular ABL1 kinase activity detection reagent kit and application thereof
A detection kit and kinase activity technology, applied in the field of enzyme activity detection, can solve the problems of long cycle, difficult expression, difficult CML treatment, etc., and achieve the effect of simple operation
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Embodiment 1
[0030] An embodiment of the extracellular ABL1 kinase activity detection kit of the present invention comprises the following components:
[0031] (1) ATP;
[0032] (2) ABL1 substrate polypeptide, the amino acid sequence of which is: EAIYAAPFAKKK (SEQ ID NO.1);
[0033] (3) ABL1 protein fragment sequence 1, its amino acid sequence is as follows:
[0034] EYLLSSGINGSFLVERSESSPGQRSISLRYEGRVYHYRINTASDGKLVSSESRFNTLAELVHHHSTVADGLITTLHYPAPKRNKPTVYGVSPNYDKWEMERTDITMKHKLGGGQYGEVYEGVWKKYSLTVAVKTLKEDTMEVDDFLKEAAVMKEIKHPNLVQLLGVCTREPPFYIITEFMTYGNLLDYLRECNRQEVNAVVLLYMATQISSAMEYLEKKNFIHRDLAARNCLVGENHLVKVADFGLSRMTGDTYTAHAGAKFPIKWTAPESLAYNKFSIKSDVWAFGVLLWEIATYGMSPYPGIDLSQVYELLEKDYRMERRPEGCPEKVYELMRACWQWNPSDRPSFAEIHQAFETMFQESSISDEVEKELGKQGVRGAVSTLLQAPELPTKTRSRRAAEHRDTTDVPEMPHSK(SEQ ID NO.2);
[0035] (4) ABL1 protein fragment sequence 2, its amino acid sequence is as follows:
[0036] MGSSHHHHHHSSGLVPRGSHMGPSENDPNLFVALYDFVASGDNTLSITKGEKLRVLGYNHNGEWCEAQTKNGQGWVPSNYITPVNSLEKHSWYHGPVSRNAAEYLLS...
Embodiment 2 Embodiment 1
[0038] Preparation of ABL1 protein fragment sequences 1 and 2 in Example 2 Example 1
[0039] 1) Plasmid transformation and shake flask culture
[0040] 1: Add 1 μg of pET-28a(+) and Kan plasmid expressing PTK domain to 100 μl Arctic Expression (DE3) competent bacteria, and place on ice for 20 minutes;
[0041] 2: Heat shock at 42°C for 90 sec, quickly place on ice for 3 min; add 600 μl LB culture solution;
[0042] 3: Shake at 220 rpm for 1 hour at 37°C, take 200 μl of the bacterial solution and spread it on an LB plate containing 50 μg / ml Kan, and incubate overnight at 37°C;
[0043] 4: The next morning, pick the monoclonal on the plate and inoculate it into a test tube containing 50 μg / ml Kan in 4ml LB medium, culture at 37°C with shaking at 220rpm until about 1:00 p.m., and the OD is about 0.6;
[0044] 5: According to the ratio of 1:250, inoculate in 1L LB culture medium of 100μg / ml Kan, shake at 220rpm at 37°C until the cell OD 600 0.5-0.6 (about 3h);
[0045] 6: Add...
Embodiment 3
[0062] Example 3 Using the kit of Example 1 to detect the effect of berberine inhibiting ABL1 kinase
[0063] The detection method that berberine inhibits ABL1 kinase activity comprises the following reaction steps:
[0064] 1. Add 1 μL berberine to each well of the drug treatment group successively, the concentration of berberine is (0.2, 4, 8, 10 μmol / L), 1uL ABL1 kinase substrate polypeptide (30ng EAIYAAPFAKKK) and 5uL ATP (2 μmol / L) , 1uL ABL protein fragment (30ng, the corresponding polypeptide of SEQ ID NO.2 or SEQ ID NO.3);
[0065] 2. Three replicate wells were set up for each group. After incubation at 30°C for 20 min, 10 μL of the luciferase-substrate mixture was added to each well, and the chemiluminescence value was measured on a microplate reader.
[0066] According to the detected luminescence value, the corresponding standard curve was drawn, and the standard curve was as follows: Figure 4 Shown; The drug inhibitory activity result that adopts ABL1 protein f...
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