Supercharge Your Innovation With Domain-Expert AI Agents!

Recombinant TetR protein, preparation method and kit thereof, and detection method of tetracycline antibiotic residues in milk

A technology of tetracyclines and detection methods, applied in chemical instruments and methods, recombinant DNA technology, measuring devices, etc., can solve problems such as undetectable, endangering the ecological environment, poor sensitivity, etc., to reduce detection costs and improve detection Efficiency and the effect of shortening the detection time

Pending Publication Date: 2022-07-29
HEBEI AGRICULTURAL UNIV.
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The irregular use of such drugs in the process of livestock and poultry breeding has brought about a series of problems that endanger human health: drug residues in livestock and poultry products such as meat, eggs, milk, and honey exceed the standard, drug-resistant bacteria spread rapidly among humans and animals, excretion The drugs or their metabolites in the substance are harmful to the ecological environment, etc.
However, when the content of tetracyclines in the sample to be tested is very low, detection of tetracyclines by TetR protein may fail to detect or give false positive results, and the sensitivity is not good

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Recombinant TetR protein, preparation method and kit thereof, and detection method of tetracycline antibiotic residues in milk
  • Recombinant TetR protein, preparation method and kit thereof, and detection method of tetracycline antibiotic residues in milk
  • Recombinant TetR protein, preparation method and kit thereof, and detection method of tetracycline antibiotic residues in milk

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] The embodiment of the present invention provides a recombinant TetR protein, whose amino acid sequence (as shown in SEQ ID NO.1) is:

[0051] MSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY QGKLTVAKLNIDQNPGTAPK YGIRGIPTLL LFKNGEVAAT KVGALSKGQL KEFLDANLAG SGSGHMHHHH HHSSGLVPRGSGMKETAAAK FERQHMDSPD LGTDDDDKAM ADIGSMSRLD KSKVINSALE LLNEVGIEGL TTRKLAQKLGVEQPTLYWHV KNKRALLDAL AIEMLDRHHT HFCPLEGESW QDFLRNNAKS FRCALLSHRD GAKVHLGTRPTEKQYETLEN QLAFLCQQGF SLENALYALS AVEHFTLGCV LEDQEHQVAK EERETPTTDS MPPLLRQAIELFDHQGAEPA FLFGLELIIC GLEKQLKCES KLAAALEHHH HHH。

[0052] The preparation method of the recombinant TetR protein comprises the following steps:

[0053] (1) Take 1 part of pET-32a-tetR plasmid PCR product, add 99 parts of sterile water, boil at 100°C for 10 min, and take 1 part as a template; primers 5'-CGGGATCCATGTCTAGATTAGATAAAAGT-3' and 5'-CCCAAGCTTTTAACTTTCCATTTAAGT-3', Take 1 each; add Taq DNA polymerase buffer, dNTPs, Taq DNA polymerase and MgCl 2 12.5 parts of ...

Embodiment 2

[0063] The embodiment of the present invention provides a kit for detecting tetracycline antibiotic residues in milk, which specifically includes:

[0064] (1) recombinant TetR protein whose amino acid sequence is shown in SEQ ID NO.1;

[0065] (2) Horseradish peroxidase labeled tetracycline hapten, luminol, 4-(imidazol-1-yl)phenol and hydrogen peroxide.

[0066] The synthetic method of horseradish peroxidase-labeled tetracycline hapten is: under the condition of n-butylamine, use 10 μL of isobutyl chloroformate (the volume ratio of n-butylamine and isobutyl chloroformate is 6:4, v / v) Activate the carboxyl group in 5mg tetracycline hapten, and then react with 2mL of 5mg / mL horseradish peroxidase solution (Shanghai Yuanye Biotechnology Co., Ltd.) for 8-12h; dilute 1000 times when using.

Embodiment 3

[0068] The embodiment of the present invention provides a method for detecting tetracycline antibiotic residues in milk, which mainly includes the following steps:

[0069] (a) Add 100 μL of the recombinant TetR protein aqueous solution in Example 1 as a coating solution (concentration of 0.25 μg / mL) into a 96-well plate, incubate at 4°C for 12 h, and discard the coating solution;

[0070] (b) Washing with 200 μL PBST (pH 7.5, containing 0.05% v / v Tween), drying, repeating three times;

[0071] (c) Add 150 μL of 2% nonfat milk powder solution (solvent is PBST) at 37°C, 60% humidity, incubate for 1 h, discard the liquid, and wash 3 times with 200 μL PBST;

[0072](d) Add 50 μL of horseradish peroxidase-labeled tetracycline hapten to each well (the preparation method is the same as that in Example 2), then add 50 μL of tetracycline standard solution or the milk sample solution to be tested, and add PBST as a control, incubate at 37°C for 1 h, repeat step (b);

[0073] (e) Add ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to View More

Abstract

The invention discloses a recombinant TetR protein, a preparation method and a kit thereof, and a method for detecting tetracycline antibiotic residues in milk, and belongs to the technical field of animal food safety. The amino acid sequence of the recombinant TetR protein provided by the invention is as shown in SEQ ID NO.1. The recombinant TetR protein has good affinity with tetracycline drugs, has very high sensitivity in detection of tetracycline drugs, and can be used for residue detection of tetracycline drugs such as tetracycline, aureomycin, oxytetracycline, minocycline and the like in livestock and poultry products. The detection method for tetracycline antibiotic residues in milk provided by the invention is a direct competitive chemiluminescence detection method, can be used for rapid analysis of tetracycline drug residues in milk, and can improve the detection efficiency, shorten the detection time and reduce the detection cost.

Description

technical field [0001] The invention relates to the technical field of animal food safety, in particular to a recombinant TetR protein, a preparation method and a kit thereof, and a method for detecting tetracycline antibiotic residues in milk. Background technique [0002] As a class of broad-spectrum antibiotics, tetracyclines are widely used to treat a variety of pathogenic microbial infectious diseases in humans and animals. At present, the commonly used varieties are: tetracycline, oxytetracycline, chlortetracycline, doxycycline, mino Cyclidine, tigecycline, etc. The irregular use of such drugs in livestock and poultry breeding has brought a series of problems that endanger human health: drug residues in meat, eggs, milk, honey and other livestock and poultry products exceed the standard, drug-resistant bacteria spread rapidly between humans and animals, excretion The drugs or their metabolites in the body endanger the ecological environment, etc. Among them, the resi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70G01N33/543G01N33/535G01N21/76G01N33/68
CPCC07K14/245C12N15/70G01N33/543G01N33/535G01N21/76G01N33/68C07K2319/21C12N2800/101
Inventor 刘静王建平王鸽夏婉秋
Owner HEBEI AGRICULTURAL UNIV.
Features
  • R&D
  • Intellectual Property
  • Life Sciences
  • Materials
  • Tech Scout
Why Patsnap Eureka
  • Unparalleled Data Quality
  • Higher Quality Content
  • 60% Fewer Hallucinations
Social media
Patsnap Eureka Blog
Learn More

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2025 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com