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Progesterone whole-cell biosensor as well as preparation method and application thereof

A biosensor and progesterone technology, applied in the field of bioengineering, can solve the problem of high detection cost and achieve the effect of rapid detection

Pending Publication Date: 2022-05-17
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a progesterone whole-cell biosensor and its preparation method and application, so as to solve the problems of high detection cost, special instruments and professional analysts in the detection of progesterone in the prior art

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  • Progesterone whole-cell biosensor as well as preparation method and application thereof
  • Progesterone whole-cell biosensor as well as preparation method and application thereof
  • Progesterone whole-cell biosensor as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Construction of progesterone whole-cell biosensor

[0035] First, seven non-naturally occurring transcriptional regulators were de novo designed. The transcriptional regulator DLA is composed of prokaryotic protein DNA-binding domain DBD, truncated human progesterone receptor B (PRB) and prokaryotic protein activation domain AD. composition. Among them, DBD, PRB and AD are lexA, PRB (682-933aa) and B42, respectively; there are also 6 truncated PRBs: 582-933aa, 482-933aa, 382-933aa, 282-933aa, and 20-933aa. In this example, PRB (682-933aa) was used to design and synthesize transcriptional regulators.

[0036] Then, a reliable structural model of DLA was established based on theoretical modeling, and molecular dynamics was used to simulate the conformational changes of DLA after progesterone binding, and the distance between the three-dimensional geometric centers of domains lexA and B42 was calculated. Make sure its center distance is around 3.5nm.

[0037]...

Embodiment 2

[0042] Example 2: Construction of progesterone whole-cell biosensor

[0043] First, seven non-naturally occurring transcriptional regulators were de novo designed. The transcriptional regulator DLA is composed of prokaryotic protein DNA-binding domain DBD, truncated human progesterone receptor B (PRB) and prokaryotic protein activation domain AD. composition. Among them, DBD, PRB and AD are lexA, PRB (20-933aa) and B42 respectively; there are also 6 truncated PRBs: 582-933aa, 482-933aa, 382-933aa, 282-933aa, and 682-933aa. In this example, PRB (20-933aa) was used to design and synthesize transcriptional regulators.

[0044] Then, a reliable structural model of DLA was established based on theoretical modeling, and molecular dynamics was used to simulate the conformational changes of DLA after progesterone binding, and the distance between the three-dimensional geometric centers of domains lexA and B42 was calculated. Make sure its center distance is around 3.5nm.

[0045] N...

Embodiment 3

[0050] Embodiment 3: the preparation of kit

[0051] Store the yeast after freeze-drying and make a test kit for detecting progesterone, which includes freeze-dried yeast, 20-fold concentration of YPD, 10 mg / L standard progesterone, 10% sodium dodecyl sulfate (SDS) and 1.5 mL test tube.

[0052] The kit is used to detect progesterone-containing samples, such as medical samples, progesterone-contaminated samples in the environment, and reaction solutions containing progesterone in the steroid hormone industry.

[0053] First, use sodium hydroxide or hydrochloric acid to adjust the pH of the liquid between 5-8;

[0054] Then, a simple rehydration of the yeast at 30 degrees for 0.5 hours was carried out, and then the yeast and the reaction solution sample from the steroid hormone industry were mixed in a test tube and incubated on a heating block at 35 degrees for 2 hours for detection;

[0055] Then, use a microplate reader to read the luminescence intensity of the sample;

...

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Abstract

The invention provides a progesterone whole-cell biosensor as well as a preparation method and application thereof. The invention designs a design strategy of a non-natural transcription factor DLA based on a tiny conformation change of a ligand binding domain LBD after a target molecule is combined with a receptor of the target molecule. A simulation result shows that a pronucleus LexA, a truncated LBD from a progestational hormone receptor and a pronucleus B42 jointly form the DLA with a transcription factor function. In order to verify the effectiveness of the design, DLA is used as a transcription activating factor for controlling green fluorescent protein transcription, and a progesterone specific whole-cell biosensor is successfully constructed, and the sensitivity index (EC50) of the progesterone specific whole-cell biosensor is 27 [mu] g / L and reaches the clinical detection level. According to the present invention, the scientific, accurate and rapid progesterone content detection method is established, and the method is particularly suitable for direct detection of trace progesterone in the sample, and is the progesterone microbiological detection method with the progesterone microbiological detection method with the progesterone microbiological detection prospect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and more specifically relates to a progesterone whole-cell biosensor and a preparation method and application thereof. Background technique [0002] Progesterone (progesterone), also known as progesterone hormone and luteinizing hormone, is the main biologically active progesterone secreted by the ovary; it is a small steroidal molecule with a molecular weight of 314.5 (C 21 h 30 o 2 ). Before ovulation, the progesterone hormone produced by the ovary is 2-3 mg per day, and after ovulation, it rises to 20-30 mg per day. Progesterone is mainly secreted by the corpus luteum in the ovary. Progesterone can protect the endometrium of women. During pregnancy, progesterone hormone can provide support and protection for the early growth and development of the fetus, and can play a certain calming effect on the uterus. In addition, the relationship between progesterone and estrogen is inseparabl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/85C12N15/65C12N1/19C12R1/865
CPCC07K14/721C12N15/85C12N15/65C07K2319/80
Inventor 王风清刘坤魏东芝
Owner EAST CHINA UNIV OF SCI & TECH
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