CN106053829B - It is a kind of based on measure p38MAPK protein expressions to detect the method for zebra fish M3 receptor proteins - Google Patents
It is a kind of based on measure p38MAPK protein expressions to detect the method for zebra fish M3 receptor proteins Download PDFInfo
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Abstract
It is a kind of based on measure p38MAPK protein expressions to detect the method for zebra fish M3 receptor proteins, comprise the following steps：(1) preparation of zebra fish brain tissue protein sample；(2) sds gel electrophoresis；(3) transferring film；(4) close；(5) primary antibody, secondary antibody are incubated；(6) chromogenic reaction；(7) M3 acceptor result of variations is calculated.No specific aim antibody is limited by instant invention overcomes the detection of traditional zebra fish M3 acceptors and can not use the defect of protein immunoblotting technology, it is proposed first using p38MAPK phosphorylation levels so as to determine zebra fish M3 receptor protein changes of contents, this method high sensitivity, stability are good, do not need complex device；While simple and quick, specific height of the invention, cost are low, the present invention studies the inherent mechanism of action of cholinergic system in terms of molecule, in conjunction with the research in terms of zebra fish Behavioral change, can finally realize the real-time online assessment to water quality with monitoring.
Description
Technical field
The invention belongs to Physiological Ecology technical field, and in particular to one kind is based on protein immunoblotting technology for detection spot
The method of horse fish M3 receptor proteins.
Background technology
In recent years, with the rapid development of economy, water pollution problems becomes increasingly conspicuous, have a strong impact on ecological environment balance and
It is stable.Zebra fish is a kind of tropica minor fresh-water fishes, and in recent years, numerous researchs show that zebra fish is applied to ecotoxicology research
In have its unique advantage, zebra fish has been increasingly becoming the new lover of Environmental Toxicological scholars, using zebra fish to water environment pollution
Thing carries out fast slowdown monitoring turns into the focus studied at present.
At present, people are generally according to the zebra fish death rate, abnormal rate, body length, body weight, body performance figure (BMI) etc.
It is measured, but the biological pathology damage of zebra fish or death often more lag, it is difficult to play the effect of fast slowdown monitoring.
In recent years, have increasing research point out fish to the adaptive response of pollutant since neuroendocrine activity change, and
Acceptor of the M3 acceptors (one of muscarinic receptor subtypes) as neurotransmitter acetylcholine, its protein content change necessarily by
Pollutant effects.Therefore, to the achievable fast slowdown monitoring to water environment pollution thing of measure of zebra fish M3 receptor protein activities.
However, the measure of M3 receptor protein activities is concentrated mainly at present on rat, mouse, electric ray these species, side
Method mainly radiates binding analysis method, protein immunoblotting technology (Western Blotting), ELISA etc., wherein,
Western Blotting are to detect protein properties, expression and a kind of the most frequently used, ripe method of distribution, and this method is not only
Target protein can be detected from protein mixture, can be with protein in qualitative or sxemiquantitative determination cell or tissue
Expression.Due to that must use the antibody with destination protein specific binding in this method, and there has been no for detecting at present
The antibody of zebra fish M3 acceptors, and because M3 receptor proteins have a greatest differences between tissue and kind, commercially available its
His M3 receptor antibodies such as people M3 receptor antibodies, mouse M3 receptor antibodies etc. and zebra fish M3 receptor binding extreme differences, this causes egg
White matter immunoblot assay may not apply to the detection of zebra fish M3 acceptors.
The content of the invention
For above-mentioned the deficiencies in the prior art, inventor has found that p38MAPK signal transduction pathways are primarily involved in by studying
It is extracellular stress caused by intracellular multiple protein kinases chain reaction, and then influence rna transcription and albumen synthesis and thin
The biological functions such as cellular surface expression of receptor, wherein, molecular switch of the phosphorylation as a signal transduction, in different paths,
As metabolism, neuronal signals transduction, cell division etc. control protein active, and when being contaminated thing pollution, p38MAPK
Protein expression (phosphorylation) changes with Regulate signal path especially nerve signal path, can pass through p38MAPK eggs with this
White expression change can determine the change of zebra fish M3 acceptors under the various concentrations exposure of different pollutants.
Specifically, the present invention relates to following technical scheme：
Application of the p38MAPK protein expressions in zebra fish M3 receptor proteins are detected.
It is a kind of based on measure p38MAPK protein expressions to detect the method for zebra fish M3 receptor proteins, comprise the following steps：
(1) preparation of zebra fish brain tissue protein sample；(2) sds gel electrophoresis；(3) transferring film；(4) close；(5) one
Anti-, secondary antibody is incubated；(6) chromogenic reaction；(7) M3 acceptor result of variations is calculated.
Preferably, the mass fraction of separation gel is 10% in the sds gel electrophoresis, and the mass fraction for concentrating glue is 5%；
Preferably, the closing concretely comprises the following steps：The film to be taken a turn for the better in step (3) is placed in 5% skimmed milk power, sealed
It is 1h to close the time；
Preferably, the primary antibody is the rabbit-anti P38/ phosphorylation P38 antibody for p38MAPK acceptors；
Preferably, the secondary antibody is the goat anti-rabbit igg marked for the HRP of p38MAPK acceptors；
It is further preferred that the dilute solution of the primary antibody, secondary antibody is 5% skimmed milk power；
Specifically, it is a kind of based on measure p38MAPK protein expressions to detect the method for zebra fish M3 receptor proteins, including with
Lower step：
(1) it is prepared by protein sample extraction：Zebra fish brain tissue is taken, cell pyrolysis liquid is added and takes supernatant, is transferred to new pre-
It is placed in cold microcentrifugal tube on ice, as albumen sample, abandons precipitation, it is dense to determine albumen with Bradford Protein Assays
Degree；
(2) sds gel electrophoresis：Prepare running gel, carry out SDS-PAGE, the matter of separation gel in the sds gel electrophoresis
It is 10% to measure fraction, and the mass fraction for concentrating glue is 5%, and electrophoresis process treatment temperature is 10 DEG C；
(3) transferring film：Protein is transferred on nitrocellulose filter from gel, keeping temperature is 4 DEG C in During migration；
(4) close：It is put into 5% skimmed milk power and closes 1h, keeps not having the combination field of antibody on film in closed process
Institute, place is set to be in saturation state；
(5) primary antibody, secondary antibody are incubated：Primary antibody is diluted with 5% skimmed milk power, the film closed is put into primary antibody dilute solution
In, it is incubated overnight in 4 DEG C of incubation boxes, next day takes out incubation at room temperature 1h in refrigerator；Then film is placed into and 3 is cleaned in TBST
It is secondary, each 15min；Period dilutes secondary antibody with 5% skimmed milk power, and cleaned film, which is put into secondary antibody, is incubated 2h, Ran Houyong
TBST washes film 3 times, each 15min.
(6) chromogenic reaction：Chemiluminescence agent (ECL) detection NC films on destination protein, then in darkroom with film to X-ray
Exposure, is scanned, and records experimental result.
(7) M3 acceptor result of variations is calculated：The experimental result drawn by step (6) calculates zebra fish M3 acceptors and changed.
Assessed and the application in monitoring in water quality real-time online the invention also discloses the above method.
Reaction mechanism of the present invention：P38MAPK signal transduction pathways be primarily involved in it is extracellular stress caused by it is intracellular a variety of
The chain reaction of protein kinase, and then rna transcription and the biological function such as albumen synthesis and cell surface receptor expression are influenceed,
Wherein, molecular switch of the p38MAPK phosphorylations as a signal transduction, in different paths, such as metabolism, neuronal signals transduction,
Cell division etc. controls protein active, and when being contaminated thing pollution, p38MAPK occur Expression of phosphorylated change with
Regulate signal path especially nerve signal path, therefore different dirts can be determined by p38MAPK phosphorylation levels with this
Contaminate the change of the lower M3 acceptors of thing various concentrations exposure.And p38MAPK is as an evolution conservative protein, the difference between different plant species
Property it is not notable, therefore the antibody to being directed to the design of other species on the market also has a good specific binding, realizes of the invention
It is changed so as to determine zebra fish M3 receptor proteins by using protein immunoblotting technology for detection p38MAPK phosphorylation levels
Purpose.
Beneficial effects of the present invention are：
(1) it is limited by no specific aim antibody instant invention overcomes the detection of traditional zebra fish M3 acceptors and protein can not be used
The defect of immunoblot assay, propose first using p38MAPK phosphorylation levels so as to determine zebra fish M3 receptor protein contents
Change, this method high sensitivity, stability are good, do not need complex device；
(2) simple and quick, specific height of the invention, cost are low, and the present invention studies cholinergic system in terms of molecule
Inherent mechanism of action, in conjunction with the research in terms of zebra fish Behavioral change, can finally realize and the real-time online of water quality is commented
Estimate and monitor.
Brief description of the drawings
Fig. 1 is experimental example Western Blot result figures；
Fig. 2 is biological behavior model step by step.
Embodiment
Embodiment
(1) it is prepared by protein sample extraction：Zebra fish brain tissue is taken, cell pyrolysis liquid is added and takes supernatant, is transferred to new pre-
It is placed in cold microcentrifugal tube on ice, as albumen sample, abandons precipitation, it is dense to determine albumen with Bradford Protein Assays
Degree；
(2) sds gel electrophoresis：Prepare running gel, carry out SDS-PAGE, the matter of separation gel in the sds gel electrophoresis
It is 10% to measure fraction, and the mass fraction for concentrating glue is 5%, and electrophoresis process treatment temperature is 10 DEG C；
(3) transferring film：Protein is transferred on nitrocellulose filter from gel, keeping temperature is 4 DEG C in During migration；
(4) close：It is put into 5% skimmed milk power and closes 1h, keeps not having the combination field of antibody on film in closed process
Institute, place is set to be in saturation state；
(5) primary antibody, secondary antibody are incubated：Primary antibody is diluted with 5% skimmed milk power, the film closed is put into primary antibody dilute solution
In, 4 DEG C are incubated in box overnight, and next day takes out incubation at room temperature 1h in refrigerator；Then film is placed into TBST and cleaned 3 times,
Each 15min；Period dilutes secondary antibody with 5% skimmed milk power, and cleaned film, which is put into secondary antibody, is incubated 2h, is then washed with TBST
Film 3 times, each 15min.
(6) chromogenic reaction：Chemiluminescence agent (ECL) detection NC films on destination protein, then in darkroom with film to X-ray
Exposure, is scanned, and records experimental result.
(7) result of calculation：The experimental result drawn by step (6) calculates zebra fish M3 acceptors and changed.
Wherein, the primary antibody is the rabbit-anti P38/ phosphorylation P38 antibody for p38MAPK acceptors；The secondary antibody be for
The goat anti-rabbit igg of the HRP marks of p38MAPK acceptors.
Western Blot methods determine decis to zebra fish M3 receptor influences experimental examples
The one kind of decis as pyrethrin pesticide, it has the function that to significantly inhibit zebra fish M3 acceptors, while agent
Graded effect is obvious.
1. exposure experiment and the acquisition of tissue homogenate
The LC50-48h of decis is determined, using this concentration as a toxic unit (1TU), 0.5TU, 1TU, 2TU are set
Three concentration gradients, 12h continuous samplings.
2. immunoblotting determines the content of M3 acceptors
(1) it is prepared by protein sample extraction：Zebra fish brain tissue is taken, cell pyrolysis liquid is added and takes supernatant, is transferred to new pre-
It is placed in cold microcentrifugal tube on ice, as albumen sample, abandons precipitation, it is dense to determine albumen with Bradford Protein Assays
Degree；
(2) sds gel electrophoresis：Prepare running gel, carry out SDS-PAGE, the matter of separation gel in the sds gel electrophoresis
It is 10% to measure fraction, and the mass fraction for concentrating glue is 5%, and electrophoresis process treatment temperature is 10 DEG C；
(3) transferring film：Protein is transferred on nitrocellulose filter from gel, keeping temperature is 4 DEG C in During migration；
(4) close：It is put into 5% skimmed milk power and closes 1h, keeps not having the combination field of antibody on film in closed process
Institute, place is set to be in saturation state；
(5) primary antibody, secondary antibody are incubated：Primary antibody is diluted with 5% skimmed milk power, the film closed is put into primary antibody dilute solution
In, 4 DEG C are incubated in box overnight, and next day takes out incubation at room temperature 1h in refrigerator；Then film is placed into TBST and cleaned 3 times,
Each 15min；Period dilutes secondary antibody with 5% skimmed milk power, and cleaned film, which is put into secondary antibody, is incubated 2h, is then washed with TBST
Film 3 times, each 15min.
(6) chromogenic reaction：Chemiluminescence agent (ECL) detection NC films on destination protein, then in darkroom with film to X-ray
Exposure, is scanned, and records experimental result.
(7) result of calculation：The experimental result drawn by step (6) calculates zebra fish M3 acceptors and changed.
Wherein, the primary antibody is the rabbit-anti P38/ phosphorylation P38 antibody for p38MAPK acceptors；The secondary antibody be for
The goat anti-rabbit igg of the HRP marks of p38MAPK acceptors.
Decis is to zebra fish brain p38MAPK depression effect as shown in figure 1, detection p38MAPK Western
Blot results show, with the increase of decis drug concentration, p38MAPK band becomes narrow gradually, and show with exposure medicine
The increase of thing concentration, p38MAPK expression quantity gradually reduce, so, by the levels of p38MAPK phosphorylations can medicine not
The change of lower zebra fish brain receptors is exposed with concentration.
Although above-mentioned the embodiment of the present invention is described with reference to accompanying drawing, model not is protected to the present invention
The limitation enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not
Need to pay various modifications or deformation that creative work can make still within protection scope of the present invention.
Claims (2)
1. a kind of based on p38MAPK protein phosphorylations horizontal expression is determined to detect the method for zebra fish M3 receptor proteins, it is special
Sign is, comprises the following steps：
（1）It is prepared by protein sample extraction：Zebra fish brain tissue is taken, cell pyrolysis liquid is added and takes supernatant, be transferred to new precooling
It is placed in microcentrifugal tube on ice, as albumen sample, abandons precipitation, protein concentration is determined with Bradford Protein Assays；
（2）Sds gel electrophoresis：Prepare running gel, carry out SDS-PAGE, the quality point of separation gel in the sds gel electrophoresis
Number is 10%, and the mass fraction for concentrating glue is 5%, and electrophoresis process treatment temperature is 10 DEG C；
（3）Transferring film：Protein is transferred on nitrocellulose filter from gel, keeping temperature is 4 DEG C in During migration；
（4）Closing：It is put into 5% skimmed milk power and closes 1h, keeps not having the combination place of antibody on film in closed process, make
Place is in saturation state；
（5）Primary antibody, secondary antibody are incubated：Primary antibody is diluted with 5% skimmed milk power, the film closed is put into primary antibody dilute solution, 4
DEG C be incubated box in overnight, next day taken out in refrigerator incubation at room temperature 1h；Then film is placed into and cleans 3 times in TBST, every time
15min；Period dilutes secondary antibody with 5% skimmed milk power, and cleaned film, which is put into secondary antibody, is incubated 2h, then washes film 3 with TBST
It is secondary, each 15min；
（6）Chromogenic reaction：Destination protein on chemiluminescence agent detection NC films, is then exposed to X-ray with film in darkroom, carried out
Scanning, record experimental result；
（7）Result of calculation：Pass through step（6）The experimental result drawn calculates the change of zebra fish M3 acceptors；
The primary antibody is the rabbit-anti phospho p38 MAPK antibody for p38MAPK；
The secondary antibody is the goat anti-rabbit igg marked for p38MAPK HRP.
2. claim 1 methods described is assessed and the application in monitoring in water quality real-time online.
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| CN113501875B (en) * | 2021-07-16 | 2022-10-18 | 四川大学华西医院 | Nano antibody specifically binding to protein kinase p38 gamma, nucleic acid, expression vector, host cell and application thereof |
Citations (1)
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| CN104931709A (en) * | 2015-06-10 | 2015-09-23 | 山东师范大学 | Method for quantitative detection of zebra fish M3a receptor proteins by use of double-antibody sandwich ELISA |
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| CN104931709A (en) * | 2015-06-10 | 2015-09-23 | 山东师范大学 | Method for quantitative detection of zebra fish M3a receptor proteins by use of double-antibody sandwich ELISA |
Non-Patent Citations (4)
| Title |
|---|
| Erk1/2- and p38 MAP kinase-dependent phosphorylation and activation of cPLA2 by m3 and m2 receptors;HUIPING ZHOU et al;《Am J Physiol Gastrointest Liver Physiol》;20030301;第284卷(第3期);第G472－G480页 * |
| Irisin Induces Angiogenesis in Human Umbilical Vein Endothelial Cells In Vitro and in Zebrafish Embryos In Vivo via Activation of the ERK Signaling Pathway;FeiWu et al;《PLOS ONE》;20150804;第10卷(第8期);第1－17页 * |
| Muscarinic receptors prevent oxidative stress-mediated apoptosis induced by domoic acid in mouse cerebellar granule cells;Gennaro Giordano et al;《J. Neurochem.》;20091231;第109卷;第525－538页 * |
| 神经抑制类农药对斑马鱼胆碱能受体的影响;刘亮;《中国优秀硕士学位论文全文数据库》;20140603;第1－52页 * |
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