A manganese ion-binding peptide, its screening method, and its affinity performance detection method

A manganese ion-binding peptide technology, which is applied in the field of manganese ion-binding peptides and their screening methods, and the detection of affinity properties, can solve unrealized problems

Active Publication Date: 2021-02-19
FUQING BRANCH OF FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, manganese pollution has not yet been realized by means of manganese ion-binding peptides.

Method used

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  • A manganese ion-binding peptide, its screening method, and its affinity performance detection method
  • A manganese ion-binding peptide, its screening method, and its affinity performance detection method
  • A manganese ion-binding peptide, its screening method, and its affinity performance detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] A screening method for manganese ion-binding peptides, comprising the following steps,

[0144] (1) Screen tetracycline-resistant Escherichia coli: add tetracycline to LB medium, cultivate Escherichia coli, and make the growth curve of Escherichia coli;

[0145] (2) Ni-NTAagarose resin to remove Ni 2+ , add Mn 2+ : Draw 0.3mL of Ni-NTAagarose resin into a sterile container, add pre-treated TBS buffer and mix well, centrifuge at 3500r / min to remove the supernatant, repeat 3 times, use pH8.0, concentration of 0.5mol / L EDTA Wash 3 times; in the same way, after washing 3 times with 0.1% TBST, add 1mL of MnCl with a concentration of 0.2g / L 2 , stored in a refrigerator at 2°C overnight, washed 3 times with TBS, and then washed 3 times with 0.1% TBST to obtain a colorless resin;

[0146] (3) Add the original library of phage random dodecapeptide library and incubate: Take the colorless resin obtained in (2) and add 0.5mL of BSA, 2°C, 150°C for sealing and slow shaking for 1...

Embodiment 2

[0157] A screening method for manganese ion-binding peptides, comprising the following steps,

[0158] (1) Screen tetracycline-resistant Escherichia coli: add tetracycline to LB medium, cultivate Escherichia coli, and make the growth curve of Escherichia coli;

[0159] (2) Ni-NTAagarose resin to remove Ni 2+ , add Mn 2+ : Draw 0.8mL of Ni-NTAagarose resin into a sterile container, add pre-treated TBS buffer and mix well, centrifuge at 4500r / min to remove the supernatant, repeat 3 times, use EDTA with pH 8.0 and a concentration of 0.5mol / L Wash 3 times; in the same way, after washing 3 times with 0.5% TBST, add 3 mL of MnCl with a concentration of 0.2 g / L 2 , stored in a 4°C refrigerator overnight, washed three times with TBS, and then washed three times with 0.5% TBST to obtain a colorless resin;

[0160] (3) Add the original library of phage random dodecapeptide library and incubate: take the colorless resin obtained in (2) and add 1mL of BSA, seal at 4°C at 200°C for 3h, ...

Embodiment 3

[0171] A screening method for manganese ion-binding peptides, comprising the following steps,

[0172] 1. Preparation of growth curve of host strain ER2738

[0173] Tetracycline was added to LB medium to cultivate Escherichia coli, namely the host strain ER2738.

[0174] (1) The bacterial strain ER2738 containing 50% glycerol buffer preserved at -80°C was picked up with an inoculation needle and streaked on the LB-tet plate, cultured overnight at 37°C in the dark.

[0175] (2) Pick a single colony of ER2738 on the plate and place it in 20mL (250mL Erlenmeyer flask) liquid LB-tet medium, culture overnight at 37°C on a shaker at 180rpm;

[0176] (3) Dilute the above bacterial solution into fresh 100mL LB-tet liquid medium according to the ratio of 1:100, take 3mL bacterial solution every 1h to measure OD 600 The absorbance values ​​are plotted as a curve.

[0177] 2. Resin treatment

[0178] (1) Removal of Ni 2+

[0179] Prepare sterile centrifuge tubes in advance, pipette...

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Abstract

The invention belongs to the field of environmental protection, and specifically relates to a manganese ion-binding peptide, which has high affinity with manganese ions and is applied to the bioremediation of manganese ions in environmental metal pollution. The invention provides a screening method for manganese ion-binding peptides. The peptide with the strongest specific binding to manganese ions is screened out by displaying a phage peptide library; Resistant Escherichia coli ER2738 acts as a phage host cell by removing Ni 2+ The elutriation of the Ni-NTA agarose resin can screen out phage polypeptides with specific affinity for manganese ions. The invention provides a detection method for the affinity performance of manganese ion-binding peptides, which can quickly and accurately compare the affinity of different phage polypeptides to manganese ions by enzyme-linked immunoassay, and use this method to quickly apply manganese ion-binding peptides to Treatment of environmental manganese pollution.

Description

technical field [0001] The invention belongs to the field of environmental protection, and in particular relates to a manganese ion-binding peptide, a screening method thereof, and a detection method for affinity performance. Background technique [0002] Manganese ion-binding peptide is a metal-binding peptide. In modern science and technology, metal-binding peptides are used in the treatment of heavy metal pollution in the environment. Due to their obvious effects, high efficiency, and low cost, they gradually replace physical repair and chemical repair, and become the current treatment of heavy metals in the environment. One of the indispensable ways of pollution. Metal ion-binding peptides form complexes with metal ions in the environment to reduce, enrich or eliminate the toxicity of metal ions to biological cells. [0003] Manganese pollution refers to the pollution of manganese to the environment. More than 500 micrograms per cubic meter in the air can cause manganes...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08C40B30/04G01N33/68A62D3/33A62D101/43
CPCA62D3/33A62D2101/43C07K7/08C12N15/1037G01N33/68G01N2410/00
Inventor 王艳君董倩曹智张文森
Owner FUQING BRANCH OF FUJIAN NORMAL UNIV
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