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Gene for degrading tetracycline antibiotics and enzyme protein encoded by gene

A technology of tetracyclines and antibiotics, applied in the field of genes and their encoded enzyme proteins, can solve problems such as tetracycline antibiotic residues, reduce the risk of drug-resistant gene transmission, and avoid threats to human health and safety

Inactive Publication Date: 2019-10-08
广东实验中学
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to overcome the deficiencies of the above-mentioned prior art, the present invention proposes a gene for degrading tetracycline antibiotics and its encoded enzyme protein, aiming to find a new direction for degrading tetracycline antibiotics from the gene level and protein level, and to solve the problem of tetracycline antibiotics in the environment. Provide a new and safe solution to the residue problem of tetracycline antibiotics, thus avoiding the biological risk of the transmission of tetracycline antibiotic resistance genes

Method used

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  • Gene for degrading tetracycline antibiotics and enzyme protein encoded by gene
  • Gene for degrading tetracycline antibiotics and enzyme protein encoded by gene
  • Gene for degrading tetracycline antibiotics and enzyme protein encoded by gene

Examples

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Effect test

Embodiment 1

[0021] Example 1 Obtaining of genes for degrading tetracycline antibiotics and their encoded enzyme proteins

[0022] Feces from a pig farm in Conghua, Guangzhou were used as experimental samples, diluted with normal saline, spread on LB solid medium (Guangdong Huankai Biotechnology Co., Ltd.), incubated at 37 °C for 12 h, and selected all the different forms of The single colony is purified, and the purified single colony is numbered and preserved. All the isolated strains were diluted with MH agar (Guangdong Huankai Biotechnology Co., Ltd.) in accordance with the guidelines of the Clinical Experimental Standardization Committee (Clinical and Laboratory Institute, CLSI). , doxycycline, minocycline) to screen for strains with tetracycline-resistant phenotypes. Among them, tetracyclines were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0023] The above-mentioned strains with tetracycline-resistant phenotype were inoculated in 4 mL LB test tube broth (Guangdong Hua...

Embodiment 2

[0037] Example 2 Degradation of tetracycline antibiotic genes ( tde ) clone

[0038] in order to tde The gene was cloned and expressed, and pET32a was selected as the protein expression vector. First, the SnapGene software was used to compare the retrieved tde Gene (nucleotide sequence as shown in SEQ ID NO: 1) for primer design to amplify the full-length sequence of the target gene, and at the same time add Bam HI and Eco RI has two enzyme cutting sites and protective bases to ensure the positive expression of the gene. The upstream primer of the designed primer pair is shown in SEQ ID NO: 2, and the downstream primer is shown in SEQ ID NO: 3. The designed primers were synthesized by Guangzhou Qingke Biotechnology Co., Ltd.

[0039] Using the genome of the strain HNS2-5-2 as a template and the above primer pair as primers, PCR amplification was performed using Ex-Taq DNA polymerase (purchased from Treasure Bioengineering (Dalian) Co., Ltd.). The PCR amplification pro...

Embodiment 3

[0040] Example 3 Enzyme protein gene for degrading tetracycline antibiotics ( tde )expression

[0041] The protein expression engineering strain BL21-pET32a- tde Inoculated in 4 ml LB liquid medium (containing 50 mg / L ampicillin), and cultured with shaking at 37 °C for 8 h; added to 100 mL LB broth (containing 50 mg / L ampicillin), cultured with shaking at 37°C until OD 600 The value is 0.4-0.6; add the inducer isopropyl β-D-Thiogalactoside [Isopropyl β-D-Thiogalactoside, IPTG (Bao Bioengineering (Dalian) Co., Ltd.)] to a final concentration of 0.5mmol / L, shake culture at 16 °C After 16 h, the protein was purified using the HIS-tagged protein purification kit from Kangwei Century Company. The purified protein was quantitatively analyzed using the BCA protein quantification kit produced by Kangwei Century Company, and the purified protein was identified by Western blot using the one-step rapid WB kit produced by Kangwei Century Company. Determination of BL21-pET32a- by BCA...

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Abstract

The invention relates to a gene for degrading tetracycline antibiotics and an enzyme protein encoded by the gene, and belongs to the technical field of biological genetic engineering. The gene has a nucleotide sequence as shown in SEQ ID NO:1, the enzyme protein has an amino acid sequence as shown in SEQ ID NO:4, the gene and the enzyme protein encoded by the gene are expected to be applied to degradation of the tetracycline antibiotics in the environment, and a new way is provided for solving the degradation problem of the tetracycline antibiotics at the gene level and the protein level; andthe degradation of the tetracycline antibiotics in a gene or protein form is beneficial to reducing the risks of drug-resistant gene transmission in the environment, and the human health and safety threats caused by the spread of tetracycline degrading enzyme (tde) genes from engineering bacteria to pathogenic bacteria caused by the direct release of degradation strains are avoided.

Description

technical field [0001] The invention relates to a gene for degrading tetracycline antibiotics and its encoded enzyme protein, which belongs to the technical field of biological genetic engineering. Background technique [0002] Tetracycline antibiotics are the most produced and used antibiotics in my country's livestock and poultry industry. In 1999 alone, the consumption of tetracycline antibiotics in my country was as high as 9,413 tons. By 2003, the output of oxytetracycline in my country accounted for the world's total output. The output of doxycycline ranks first in the world. In 2013, the total consumption of tetracycline antibiotics in my country has reached 12,000 tons. Due to the low absorption rate, a large amount of tetracycline antibiotics enters the environment in the form of parent or active metabolites with urine and feces after taking, causing a large amount of tetracycline antibiotics to accumulate in animal manure and the surrounding environment of the farm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/11A62D3/02A62D101/28
CPCA62D3/02A62D2101/28C12N9/0073C12Y114/13008
Inventor 郑艺李盈萱朱颖琪王剑崔超月孙坚
Owner 广东实验中学
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