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Protein TetR combinable with tetracycline and coding genes and applications of protein TetR combinable with tetracycline

A gene and coding technology, which is applied to the protein TetR combined with tetracycline and its coding gene and application field, can solve the problems of destroying the balance of gastrointestinal flora, affecting human health, vitamin deficiency, etc., and achieves high sensitivity and strong specificity , to solve the effect of low rate

Inactive Publication Date: 2012-09-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Residual antibiotics in food will destroy the balance of gastrointestinal flora, and can cause long-term diarrhea, vitamin deficiency and affect the efficacy of other drugs, thereby affecting human health

Method used

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  • Protein TetR combinable with tetracycline and coding genes and applications of protein TetR combinable with tetracycline
  • Protein TetR combinable with tetracycline and coding genes and applications of protein TetR combinable with tetracycline
  • Protein TetR combinable with tetracycline and coding genes and applications of protein TetR combinable with tetracycline

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, discovery of tetracycline receptor protein and its coding gene

[0051] Escherichia coli carrying the tetracycline resistance gene (tetB) and its regulatory gene (tetR) was obtained from the Microbiological Testing Laboratory of the Livestock and Poultry Product Quality Supervision, Inspection and Testing Center of the Ministry of Agriculture, with the code number LYP-20. Escherichia coli LYP-20 strain was obtained from routine monitoring of pathogenic bacteria and isolated from Shandong breeding pig farm.

[0052] A new protein (tetracycline receptor protein) and its coding gene that can specifically bind tetracycline were found from LYP-20 bacteria.

[0053] The protein shown in Sequence 1 of the Sequence Listing was named TetR protein (expected molecular weight about 23 kDa). The gene encoding the TetR protein is named TetR gene, and its open reading frame is shown in sequence 2 of the sequence listing.

Embodiment 2

[0054] Embodiment 2, the construction of recombinant bacterium and control bacterium

[0055] 1. Construction of recombinant plasmids

[0056] 1. Artificially synthesize the double-stranded DNA molecule shown in sequence 2 of the sequence listing.

[0057] 2. Using the double-stranded DNA molecule in step 1 as a template, perform PCR amplification with the primer pair composed of F1 and R1, and recover the PCR amplification product (about 633bp).

[0058] F1: 5'-TT GCTAGC TCTAGATTAGATAAAAGTAAAG-3' (NheI restriction recognition sequence is underlined);

[0059] R1: 5'-TT CTCGAG TTAAGACCCACTTTTCACATTTA-3' (XhoI recognition sequence is underlined).

[0060] 3. Carry out double digestion of the PCR amplification product with restriction endonucleases NdeI and XhoI, and recover the digestion product.

[0061] 4. Digest the vector pET28b with restriction endonucleases NdeI and XhoI to recover the vector backbone (about 5300bp).

[0062] 5. Ligate the digested product of step...

Embodiment 3

[0067] Example 3, Preparation and Purification of TetR Protein

[0068] 1. Preparation of crude protein extract

[0069] The bacterial liquid in this step refers to all the substances in the culture vessel, including bacterial cells and supernatant.

[0070] The recombinant bacteria LYP-BLR-1, recombinant bacteria LYP-BLR-2, recombinant bacteria LYP-BLR-3, recombinant bacteria LYP-BLR-4, recombinant bacteria LYP-BLR-5, recombinant bacteria LYP-BLR-6, ​​recombinant Bacteria LYP-BLR-7, recombinant bacteria LYP-BLR-8 and recombinant bacteria BL21-pET28b were processed in parallel as follows:

[0071] 1. The strain was inoculated into 2 mL of LB liquid medium containing 30 μg / mL kanamycin, and cultured with shaking at 37° C. and 250 rpm for 8 hours to obtain a seed liquid.

[0072] 2. Inoculate 1mL of the seed liquid from step 1 into 200mL LB liquid medium containing 30μg / mL kanamycin, culture at 37°C with shaking at 250rpm until OD 600 =0.6; take out 1mL of the bacterial liqui...

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Abstract

The invention discloses a protein TetR combinable with tetracycline and coding genes and applications of the protein TetR combinable with tetracycline. The protein which can be specifically combined with the tetracycline is (a) or (b): (a) is a protein composed by the amino acid sequence shown by sequence 1 in the sequence list, and (b) is a protein derived from (a) by subjecting the amido acid residue sequence of the sequence 1 in the sequence list to substitution and / or deletion and / or addition of one or multiple amido acid residues and by combination with tetracycline antibiotics. Recombined escherichia coli can be obtained by leading coding genes of tetracycline receptor protein into starting escherichia coli. The invention further discloses a method for preparing the protein, namely cultivation of the recombined strain. Experiments show that the method for preparing the tetracycline receptor protein is high in yield, and problems of low yield and difficulty in preparation in the prior art are solved, so that the method is of great importance.

Description

technical field [0001] The invention relates to a protein TetR combined with tetracycline, its coding gene and application. Background technique [0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics produced by actinomycetes, including chlortetracycline, oxytetracycline, tetracycline and semi-synthetic derivatives methoxytetracycline, Doxycycline, dimethylaminocycline, etc., all contain naphthacene basic skeleton in their structures. [0003] Residual antibiotics in food will destroy the balance of gastrointestinal flora, and can cause long-term diarrhea, vitamin deficiency and affect the efficacy of other drugs, thereby affecting human health. At present, the problem of antibiotic residues has attracted widespread attention. As a necessity in people's life, the problem of antibiotic residues in milk has attracted more and more attention. In order to ensure the quality and safety of milk, in addition to strictly controlling the use of antibiotics in t...

Claims

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Application Information

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IPC IPC(8): C07K14/245C12N15/31C12N15/63C12N5/10C12N1/21C12P21/02C12R1/19
Inventor 沈建忠汪洋吴聪明曹兴元
Owner CHINA AGRI UNIV
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