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Zebrafish vitellogenin immunoblotting kit and its detection method and application

A technology of immunoblotting and original protein, which can be used in measurement devices, biological tests, material testing products, etc., can solve the problems of influence effect, poor specificity and sensitivity, etc., achieve large induction amount, simplify experimental methods, and reduce induction time. and workload effects

Inactive Publication Date: 2016-04-13
OCEAN UNIV OF CHINA
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Problems solved by technology

However, fish vitellogenin is a species-specific protein, and the vitellogenin of different fishes is quite different, and heterologous antibodies will show poor specificity and sensitivity when used for the detection of vitellogenin in other fish , affecting the detection effect

Method used

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  • Zebrafish vitellogenin immunoblotting kit and its detection method and application
  • Zebrafish vitellogenin immunoblotting kit and its detection method and application

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Embodiment Construction

[0021] The preparation of the western blot kit for detection of zebrafish vitellogenin is divided into the following steps:

[0022] (1) Induction and purification of zebrafish vitellogenin

[0023] Preparation of pure zebrafish vitellogenin: zebrafish were induced to produce vitellogenin by exposing water to 17β-estradiol, and the zebrafish were placed in a 2L glass jar, and 10 zebrafish were placed in each jar, 17β-estradiol The exposure concentration of estradiol was 200 μg / L, all the water was changed every day, and the corresponding 17β-estradiol was added again, and the feed was fed in the morning and evening, and the water temperature was 26°C; After the zebrafish is anesthetized, take it out and weigh it, add 3 times the volume of 4°C pre-cooled PBS buffer (containing 1mMPMSF, pH7.5), homogenize with a glass homogenizer under ice bath conditions, and centrifuge at 4°C at 8000g 10 minutes, collect supernatant; All supernatant that collects is used for ion-exchange chro...

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Abstract

The invention relates to a zebra fish vitellogenin immunoblotting reagent kit as well as a detection method and application thereof. The reagent kit contains a zebra fish vitellogenin pure product and a rabbit anti-zebra fish vitellogenin polyclonal antibody. The preparation method comprises the steps: producing vitellogenin by inducing the zebra fish by utilizing 17beta-estradiol, purifying the vitellogenin by virtue of a method combining the two-step chromatography and hyperfiltration, then immunizing a New Zeeland rabbit to prepare the rabbit anti-zebra fish vitellogenin polyclonal antiserum, precipitating by utilizing ammonium sulfate and carrying out chromatographic purifying by utilizing Hitrap Protein G to obtain the rabbit anti-zebra fish vitellogenin polygonal antibody. The reagent kit can be used for screening environmental estrogen substances and sensitively and conveniently qualitatively detecting the vitellogenin in zebra fish blood, integral homogenate, liver tissues and liver cell culture liquid, the minimum detection limit is 60ng / mL, and an important tool can be provided for the application of the zebra fish in the endocrine disrupter detection field.

Description

technical field [0001] The invention belongs to the field of ecological detection, and in particular relates to a zebrafish vitellogenin immunoblotting kit and a detection method and application thereof. Background technique [0002] With the continuous improvement of the degree of industrialization and the living standards of the people in our country, a large amount of industrial wastewater and domestic sewage are discharged into rivers, lakes and seas, causing serious water environmental pollution, which not only endangers the survival of wild creatures, but also seriously threatens the life of our people. healthy. Although the concentration of these pollutants in the environment is very low, they can interfere with a series of biological processes such as the synthesis, release, transportation, and metabolism of hormones in humans and animals, as well as the combination of hormones and receptors, and the expression of functions, thereby disrupting human and animal health...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/558G01N33/531
CPCG01N33/531G01N33/54306G01N33/68
Inventor 汝少国王军王蔚田华
Owner OCEAN UNIV OF CHINA
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