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A Bioluminescence-Based Method for Measuring Zebrafish M-Receptor Levels
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A bioluminescence and zebrafish technology, applied in the field of physiological ecology, can solve the problems of human harm to experimenters, expensive reagents, lack of universality, etc., and achieve the effects of low cost, high sensitivity and high specificity
Active Publication Date: 2019-05-28
SHANDONG NORMAL UNIV
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[0004] However, at present, the determination of M receptor protein activity is mainly concentrated on species such as rats, mice, and electric rays. The methods are mainly radiation binding assays, Western Blotting, ELISA, etc. These methods generally have cumbersome technical steps and time-consuming , expensive reagents, potential harm to the human body of the experimenter, etc., and there is no clear method to measure the protein content of the zebrafish M receptor, and due to the differences between the M receptor protein tissues and species , so the method for determining the M receptor protein content is not universal, so it is urgent to develop a sensitive, fast and simple method for determining the M receptor level in zebrafish
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Embodiment 1
[0030] (1) Break the zebrafish tissue, centrifuge at low temperature, take the supernatant, and place equal amounts of the supernatant in three incubation tubes A, B, and C;
[0031] (2) Convert GDP and GMP in the supernatant obtained in step (1) into GTP; more specifically, first add 50 μL of supernatant to each incubation tube, and then add pH7.5 zwitterions to each tube Buffer, MgCl 2 After that, tube A will not add any substances, tube B will add a certain amount of phosphoenolpyruvate and pyruvate kinase, tube C will add a certain amount of ATP and guanylate kinase, and the reaction mixture in each incubation tube will be uniform. Adjust to 250 μL with ultrapure water; incubate the reaction mixture and then boil the water bath to terminate the reaction, then place the incubation tube containing the reaction mixture in ice to cool, the incubation temperature is 30°C, and the time is 30min; the cooling The time is 10 minutes;
[0032] (3) Destroy the ATP and UTP in each r...
experiment example 1
[0040] 1. The experiment was divided into experimental group and control group. Six zebrafish were placed in each group, and each zebrafish was used as a sample. Samples were taken at 2h, 4h, 6h, 8h, 10h, and 12h. The experimental group was exposed to 0.01mol / L atropine (an anticholinergic drug that blocks M receptors) for 12 hours, and the control group did not add any substance.
[0041] 2. Take out the sample and add it to pre-cooled PBS, fully homogenize it in an ice bath, then centrifuge at 12,000 rpm at 4°C for 20 minutes, take the supernatant, and process it through the following steps respectively.
[0042] Three separate incubation tubes, A, B, and C, were prepared for the experimental group and the control group respectively, and were processed by the following steps:
[0043] (1) Convert GDP and GMP in the obtained zebrafish supernatant to GTP; more specifically, first add 50 μL of supernatant to each incubation tube, and then add pH7.5 zwitterions to each tube Buf...
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Abstract
The invention discloses a method for determining a zebrafish M receptor level based on a bio-luminescence method. The method comprises the following steps: preparing a zebrafish tissue supernatant, and converting GDP and GMP into GTP; destroying ATP and UTP; converting the GTP into the ATP; and detecting the ATP and processing and computing data, so that change in the zebrafish M receptor level is obtained. The method disclosed by the invention, which is used for detecting the change in the content of a zebrafish M receptor based on the bio-luminescence method, is provided for the first time, and the method is unnecessary to determine the protein content of the M receptor; the method is high in sensitivity, good in stability and free from use of complex equipment; the method disclosed by the invention is simple and convenient to operate, high in specificity and low in cost; the internal operating mechanism of a cholinergic system is researched in molecular aspect, and researches on the change of zebrafish behaviors are conducted, so that real-time online assessment and monitoring on water quality are finally achieved.
Description
technical field [0001] The invention belongs to the technical field of physiological ecology, and in particular relates to a method for measuring the level of M receptors in zebrafish based on a bioluminescent method. Background technique [0002] In recent years, with the rapid development of the economy, the problem of water pollution has become increasingly prominent, seriously affecting the balance and stability of the ecological environment. Zebrafish is a small tropical freshwater fish. In recent years, many studies have shown that zebrafish has its unique advantages in environmental toxicology research. Zebrafish has gradually become the new favorite of environmental toxicologists. Rapid monitoring of pollutants has become a research hotspot. [0003] At present, people usually measure the mortality rate, deformity rate, body length, weight, body mass index (BMI) of zebrafish, etc. However, the biological pathological damage or death of zebrafish often lags behind, a...
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