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Ochratoxin detoxification protein, encoding gene thereof and application

A technology of ochratoxin and gene, applied in the field of ochratoxin detoxification protein and its coding gene, can solve the problems of slow development of detoxification substances

Inactive Publication Date: 2017-01-25
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the research on biological detoxification focuses on the screening of detoxification microorganisms, while the research on detoxification substances develops slowly

Method used

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  • Ochratoxin detoxification protein, encoding gene thereof and application
  • Ochratoxin detoxification protein, encoding gene thereof and application
  • Ochratoxin detoxification protein, encoding gene thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1 Extraction of B.Subtilis genome

[0100] The extraction steps of B.Subtilis genome are as follows:

[0101] 1) Pick the activated B.Subtilis CW14 from LB solid medium, put it in 10mL LB liquid medium, and culture it at 37°C and 200r / min for 24h;

[0102] 2) Take 1mL of bacterial liquid in a 2mL centrifuge tube, centrifuge at 10000r / min for 5min, discard the supernatant; add 1mL of PBS buffer, resuspend the bacteria, centrifuge again, discard the supernatant;

[0103] 3) Add 500 μL of pre-cooled (stored at 4°C) ethanol, resuspend the bacteria, centrifuge at 10,000 r / min for 5 min, and discard the supernatant;

[0104] 4) Add 200 μL of PBS solution, 40 μL of 50 mg / mL lysozyme solution, pipette with a pipette gun, thoroughly suspend and precipitate, and bathe in 37°C water for 30 minutes;

[0105] 5) Add 500 μL lysate solution, 10 μL proteinase K solution, and bathe overnight at 55°C;

[0106] 6) Add 2 μL of RNase A solution and bathe in water at 37°C for 30 m...

Embodiment 2

[0114] The PCR amplification of the CPA gene sequence of embodiment 2 B.Subtilis

[0115] (1) Primer synthesis

[0116] References (Chang XJ, Wu ZD, Wu SL. Degradation of ochratoxin A by Bacillusamyloliquefaciens ASAG1. Food Additives & Contaminants: Part A, 2015, 32(4):564–571), the primer sequences were slightly changed. The primer sequences are as follows:

[0117] F: 5'-CGC GTT GAA CATCAC GAA ATG GAA-3'

[0118] R: 5'-CCC AAA CCA GCC TGT TAC CG-3'

[0119] The above primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0120] (2) PCR amplification

[0121] Using a 25 μL reaction system, using B. Subtilis genomic DNA as a template, add in sequence to a 0.5 mL Eppendorf tube:

[0122]

[0123] After mixing and centrifuging for a short time, the amplification reaction was carried out according to the following PCR conditions: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds; annealing at 60°C for 1 minute; extension at 72°...

Embodiment 3

[0127] Example 3 Construction and Identification of Recombinant Plasmid pET-28a(+) / CPA

[0128] Codon optimization and construction of recombinant plasmid pET-28a(+) / CPA ( Figure 4 ) to Beijing Aoke Dingsheng Biotechnology Co., Ltd.

[0129] Using DNAMAN to compare the optimized synthetic target fragment with the carboxypeptidase sequence in B.Amyloliquefaciens ASAG1 in NCBI ( Figure 5 ), the results showed that the similarity of the optimized base sequence reached 77.02%, and the amino acid sequence remained unchanged.

[0130] 1. Activation of Glycerol bacteria containing recombinant plasmid pET-28a(+) / CPA

[0131] Thaw the Glycerolbacterium strain containing the recombinant plasmid pET-28a(+) / CPA frozen at -80°C on ice, and flick the wall tube to mix;

[0132] 2) Take 100 μL of the bacteria and inoculate into 2 mL of fresh LB / Kan (final concentration of Kan is 25 μg / mL) liquid medium, culture overnight on a shaker at 37°C at 200 r / min, and inoculate fresh LB / Kan (final...

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Abstract

The invention relates to a microorganism protein and a gene, and particularly discloses an ochratoxin detoxification protein and an encoding gene of the ochratoxin detoxification protein. The amino acid sequence of the protein is shown as SEQ ID NO.1, and the encoding gene sequence is shown as SEQ ID NO.2. The recombinant bacteria structured by gene sequence can express the protein with ochratoxin detoxification activity.

Description

technical field [0001] The present invention relates to microbial protein and gene, in particular, relates to ochratoxin detoxification protein and its coding gene. Background technique [0002] Ochratoxins (OTs) are toxic metabolites produced by toxin-producing molds such as Aspergillus and Penicillium, and are widely distributed in food, grain and other agricultural by-products. Among them, ochratoxin A (ochratoxin A, OTA) is the most widely distributed in nature, the most toxic, and has the greatest impact on humans, animals and plants. Animal experiments have shown that OTA has multiple toxicities such as nephrotoxicity, hepatotoxicity, immunotoxicity, teratogenicity, carcinogenicity and mutagenicity, and has great potential harm to animal and human health. Therefore, preventing the generation of OTA, reducing or eliminating the OTA that has been generated is very important for food safety. [0003] At present, the detoxification research on OTA at home and abroad can ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/32C12N15/31C12N15/70C12N1/21A23L5/20C12R1/19
Inventor 梁志宏贾欣胡海宁王晓云黄昆仑
Owner CHINA AGRI UNIV
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