Recombinant yeast for detecting environmental estrogen and constructing method and use of recombinant yeast

A technology for recombining yeast and detecting the environment, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, and microbial measurement/inspection, etc., to achieve good repeatability, high sensitivity, and improved sensitivity

Inactive Publication Date: 2014-02-05
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is not conducive to mutual confirmation with some in vivo experiments, embryo teratogenic experiments, and population ecological experiments.

Method used

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  • Recombinant yeast for detecting environmental estrogen and constructing method and use of recombinant yeast
  • Recombinant yeast for detecting environmental estrogen and constructing method and use of recombinant yeast
  • Recombinant yeast for detecting environmental estrogen and constructing method and use of recombinant yeast

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Experimental program
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Effect test

Embodiment 1

[0031] A method for constructing recombinant yeast that detects environmental estrogens, comprising the following steps:

[0032] (1) Construction of expression plasmids:

[0033] Using the cDNA sequence (SEQ ID No.1) of Tang fish estrogen receptor gene TERα as a template, specific primers were designed and synthesized:

[0034] Upstream primer is 5'-A GAATTC ATGGTGATGTCTGGAGGGCA-3' (SEQ ID No. 2)

[0035] Downstream primer is 5'-A CTCGAG TTAGGTATCTGGACTTTGGCTAA-3' (SEQ ID No. 3)

[0036] EcoRI and XhoI restriction sites (underlined) were designed at both ends of the primers respectively, and the complete ORF fragment of the TERα gene was amplified by PCR. After the PCR product was purified and recovered, it was connected with the cloning vector pMD18-T to construct a recombinant plasmid named pMP18-T / TERα. pMP18-T / TERα was digested with EcoR I and Xho I, and the target fragment TERα was recovered from the gel and connected with the yeast episomal expression vector pGAD...

Embodiment 2

[0051] Using the recombinant yeast AHptERα / pr2 obtained in Example 1 to determine the content of environmental estrogen (17-β estradiol, referred to as E2) in the sample includes the following steps:

[0052] (1) Induction culture of recombinant yeast strains

[0053] Single clones of recombinant yeast AHptERα / pr2 were picked to add CuSO 4 Shaking culture in liquid SD medium, to OD 600 When the reading is 0.1-0.4, add a series of samples to be tested (DMSO solutions containing different concentrations of 17-β estradiol), shake and culture in a constant temperature shaker at 30°C for 18 hours, measure and record the OD600 value of the culture solution; Liquid SD medium does not contain leucine (Leu) and uracil (Ura), where CuSO 4 The mass volume concentration is 0.1%.

[0054] Take 1mL of the culture medium of the series of samples to be tested and put them into 1.5mL centrifuge tubes, centrifuge at 14000rpm, remove the supernatant, add 1mL of Z buffer, centrifuge again and ...

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Abstract

The invention discloses recombinant yeast for detecting environmental estrogen and a constructing method and use of the recombinant yeast. The recombinant yeast comprises a yeast expression plasmid and a yeast report plasmid, wherein the yeast expression plasmid comprises a tanichthys albonubes estrogen receptor gene TERalpha represented by the sequence shown as SEQ ID No.1, and a carrier of the yeast report plasmid is pMP206, and is linked with a tanichthys albonubes vitellogenin gene promoter 2 represented by a sequence shown as SEQ ID No.4. The tanichthys albonubes estrogen receptor is used for constructing the expression plasmid, the tanichthys albonubes vitellogenin gene promoter is linked with the report gene to construct the report plasmid. The tanichthys albonubes is sensitive to the change of the living environment, and is a wonderful fish biological marker for monitoring the environmental estrogen pollution by using a biological method, and the vitellogenin is a good biological marker for the environmental internal-secretion interfering-substance. The estrogen receptor and the vitellogenin promoter of the tanichthys albonubes are used for constructing a recombinant yeast evaluation system, so that the sensitivity can be greatly improved.

Description

technical field [0001] The invention belongs to the field of biological detection, and in particular relates to a recombinant yeast for detecting environmental estrogen and its construction method and application. Background technique [0002] Hormones are high-efficiency bioactive substances secreted by biological endocrine cells, which have the characteristics of low concentration and high potency. [0003] Environmental hormones refer to certain toxic compounds that exist in the environment, can simulate the action of natural hormones, and have a serious impact on the endocrine system of organisms. [0004] Environmental estrogen is the most important type of environmental hormones, and it is one of the main causes of reproductive disorders, reproductive system deformities, abnormal development, metabolic disorders, and increased incidence of certain malignant tumors. Due to the variety of environmental estrogens, great harm, wide distribution, and complex mechanism of a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12Q1/37C12Q1/02
Inventor 马广智陈美玲武佳韵凌源智刘琳黄儒强陈世文吴萍萍尹艳艳章凌霄黄中豪曾健辉孔繁茂朱宝君
Owner SOUTH CHINA NORMAL UNIVERSITY
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