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Method for detecting estrogen pollutions in water body based on recombined Bostrychus sinensis Vitellongenin and application

A technology of Chinese snakehead and vitellogenin, which is applied in the field of environmental pollution detection and immunology, and can solve the problems of long processing time and high cost of detection

Active Publication Date: 2020-01-24
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are mainly physical and chemical methods for the detection of environmental hormones. This type of method can mainly detect a certain chemical substance quantitatively and qualitatively, but this type of method often requires special and expensive equipment, reagents, and professional personnel. Pre-processing, processing and detection time is long and the cost is high
At present, there are no literatures and inventions related to the preparation of corresponding polyclonal and monoclonal antibodies and the use of these antibodies to monitor environmental estrogen pollution using recombinant vitellogenin

Method used

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  • Method for detecting estrogen pollutions in water body based on recombined Bostrychus sinensis Vitellongenin and application
  • Method for detecting estrogen pollutions in water body based on recombined Bostrychus sinensis Vitellongenin and application
  • Method for detecting estrogen pollutions in water body based on recombined Bostrychus sinensis Vitellongenin and application

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Experimental program
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Effect test

Embodiment 1

[0048] The preparation of recombinant Channa sinensis VTG uses the principle of genetic engineering to clone the full-length gene of Channa sinensis VTG, selects a 816bp nucleotide sequence for cloning and prokaryotic expression, clones, expresses and purifies to obtain recombinant Channa sinensis VTG. It can be seen from the SDS-PAGE figure that the recombinant expression has an obvious band at the 70KD position, which is the fusion expression region of the tag MNP (40KD) and the target protein VTG (30KD). After purification and concentration, the fusion protein is obvious, and the mixed band is relatively less, see figure 1 .

Embodiment 2

[0050] The preparation of the VTG polyclonal antibody of Channa sinensis, the preparation method comprises the following steps:

[0051] (1) Preparation of complete and incomplete Freund's adjuvants. Take an appropriate amount of recombinant VTG solution and dilute it with PBS to a VTG concentration of 0.1 mg / mL, and add an equal volume of complete Freund's adjuvant. Fully emulsify with a 1mL syringe to make complete Freund's adjuvant. An equal volume of antigen solution was fully emulsified with incomplete Freund's adjuvant to make incomplete Freund's adjuvant. Injection was performed by subcutaneous multi-point injection (0.2 mL / point).

[0052] (2) Before injecting the adjuvant, BCG was used to stimulate the popliteal fossa and paw of BalB / C mice to activate the immune function of the mice. Immunization with complete Freund's adjuvant was started one week later.

[0053] (3) For the first time, BalB / C mice were immunized with complete Freund's adjuvant of the recombinan...

Embodiment 3

[0057] Detection and Identification of Polyclonal Antibody to Vitellogenin of Channa sinensis

[0058] Application of ELISA method to detect antibody titer

[0059] The specific method is as follows:

[0060] (1) Add 100 μL VTG purified protein standard to the concave well of the microtiter plate, and incubate at 4°C for 16 hours.

[0061] (2) Pour off the residual liquid in the wells of the ELISA plate, add 300 μL PBST-20 to each well after drying, place on a shaker and shake for 5 minutes, pour out the solution after standing for 5 minutes each time, and dry in the air. Repeat 3 times.

[0062] (3) Add 250 μL of 5% skimmed milk to the wells of the microplate, seal the microplate with a parafilm, place it in a shaking incubator at full temperature, and incubate at 500 rpm for 1.5 h.

[0063] (4) Pour off the residual liquid in the concave wells of the ELISA plate, add 300 μL PBST-20 to each well, place on a shaker and shake for 5 minutes, pour out the solution after standi...

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Abstract

The invention provides a method for detecting estrogen pollutions in a water body based on recombined Bostrychus sinensis Vitellongenin and an application, relates to the field of environmental pollution detection and immunology, and discloses a method for preparing a polyclonal antibody by taking recombinant protein as an antigen based on the recombined Bostrychus sinensis Vitellongenin VTG. A fast and accurate immunoassay method for detecting the VTG is established. By means of the method, male Medaka induced by 17-beta estradiol (E2) is detected, the VTG level of male fish induced by E2 isfound to be significantly higher than that of non-induced male fish, and the method is used to identify female fish and male fish and the VTG in the female fish is found to be obviously higher than that of the male fish. The difference is extremely significant, and the technology can be used to monitor the environmental estrogen pollutions and identify the male fish and the female fish.

Description

technical field [0001] The invention belongs to the field of environmental pollution detection and immunology, and particularly relates to a detection method and application of estrogen pollution in water bodies based on recombined vitellogenin of Chinese snakehead snakehead. Background technique [0002] With the development of industrial technology, especially the rapid development of chemical synthesis technology, various chemical synthesis products are widely used in human life, and these chemical substances are difficult to degrade under natural conditions. One type of chemical substance enters the environment through water, air, and soil, and its chemical properties have similar functions to animal hormones. After entering the organism, it can compete with normal hormones, thereby disrupting the normal physiological functions of the human body, and even causing sexual development, Than chaos, some may cause cancer. The currently recognized environmental estrogens main...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/68C07K16/18C07K16/06
CPCC07K16/06C07K16/18C07K2317/35G01N33/6854G01N33/74G01N2333/4603
Inventor 周克夫余镒琦孟坤龙敏
Owner XIAMEN UNIV
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