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Genetic engineering kit for detecting environmental estrogen pollutants and application thereof

A genetic engineering and estrogen technology, applied in the field of genetic engineering kits for the detection of environmental estrogen pollutants, can solve the problems of high false positive antigen-antibody reaction, high detection threshold, high price, etc. The effect of high detection stability and low sample requirement

Inactive Publication Date: 2012-06-27
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection method of the kit is complicated, requires multi-step operation, and takes a long time; it is expensive and costly; the false positive of antigen-antibody reaction is high; the detection threshold is high, generally 500-1400ng / mL; and the scope of application of the kit Limited, can only detect vitellogenin in the serum or plasma of crucian carp and related species of fish

Method used

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  • Genetic engineering kit for detecting environmental estrogen pollutants and application thereof
  • Genetic engineering kit for detecting environmental estrogen pollutants and application thereof
  • Genetic engineering kit for detecting environmental estrogen pollutants and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Preparation of genetic engineering kit

[0033] Step 1, fusing the Ser301-Thr553 peptide segment of the human estrogen receptor α protein with the intein (Mycobacterium tuberculosis recA intein) to construct an estrogen-sensitive intein chimera;

[0034] (1) PCR amplified intein (Mycobacterium tuberculosis recA intein) fragment, primers were 5'-GGATCCtgtttcgactacagcacccg-3', 5'-AAGCTTgttgtgcaccatgacaccgt-3', purified and recovered; the fragment and plasmid pET28a were respectively used with Bam HI / After Hind III double digestion, recover the digested fragment; then use T4 DNA ligase to connect the two digested fragments, and chemically transform Escherichia coli DH5α(F-endA1 gln V44 thi-1 recA1 relA1 gyrA96 deoRnupG Φ80dlacZΔM15Δ(lacZYA-argF)U169 , hsdR17(rK-mK+), λ-), spread on LBK plates, culture overnight at 37°C; pick clones and extract plasmids, sequence and identify, positive plasmids were named pMRecA, purified and recovered, and stored at -20°C for future use. ...

Embodiment 2

[0060] Detection of Effect Levels of Various Environmental Estrogens by Genetic Engineering Kit

[0061] Using the kit obtained in Example 1, different environmental estrogen samples and sewage samples obtained from sewage treatment plants were measured, and the estrogen effect level of the samples was analyzed.

[0062] Prepare different concentrations of 17β-estradiol, bisphenol A, chrysene and 6-OH- -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 , 1×10 -9 , 1×10 -10 mg / mL) samples, stored at low temperature. The sewage sludge sample obtained from the sewage treatment plant was filtered with filter paper, and the sample to be tested was obtained by solid phase extraction. When measuring samples, inoculate estrogen-sensitive genetically engineered strains into 1mL liquid medium, shake at 37°C until OD 600 At about 0.6, add 1 μL of inducer (1:1000 dilution) and 10 μL of sample solutions with different concentrations. Shake culture at 37°C ...

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Abstract

The invention relates to a genetic engineering kit for detecting environmental estrogen pollutants and application thereof, and belongs to the technical field of detection. The kit comprises the following components in part by volume: 2*10<4> parts of LB culture medium, 1 part of isopropyl-beta-D-thiogalactoside, 20 parts of chloroform, 10 parts of solution of lauryl sodium sulfate, 20 parts of o-nitrophenyl-beta-D-galactoside and 100 parts of solution of sodium carbonate. A detection process of the genetic engineering kit is simple, convenient and quick, and results can be obtained within 2 to 3 hours; the genetic engineering kit does not have special requirements on instruments, demand quantity of samples is fewer, detection stability is high, and the detection sensitivity can reach n g / L; and the genetic engineering kit improves the analytical rate while reducing the analytical cost greatly.

Description

technical field [0001] The invention relates to a genetic engineering kit and an application method thereof in the technical field of environmental detection, in particular to a genetic engineering kit for detecting environmental estrogen pollutants and an application thereof. Background technique [0002] Environmental estrogen pollutants (Environmental Estrogens, EEs) are a class of chemical substances that persist in the environment produced by nature or human activities, and can accumulate and enrich in animals and humans through direct or indirect ways. It binds to hormone receptors, and its function is similar to that of animal endocrine hormones, which can cause nervous system disorders, endocrine disorders, immune decline, and reproductive disorders in humans and various organisms, which are directly related to the survival and development of human society and the biosphere. The fundamental security of reproduction. Therefore, EEs must be tested in an all-round way ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/78C12Q1/02C12N1/20C12R1/19
Inventor 梁如冰刘建华
Owner SHANGHAI JIAOTONG UNIV