Bacillus velezensis YtnP-homologous lactonase, gene and application thereof

A Bacillus and lactonase technology, applied in the field of genetic engineering, can solve the problems of enzyme inactivation, application limitation of QQ enzyme, etc., and achieve the effects of high enzyme activity and enzyme stability, inhibition of early proliferation, wide application prospect and value

Active Publication Date: 2021-06-15
HUAQIAO UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the sensitivity of QQ enzyme to temperature and pH, the enzyme m...

Method used

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  • Bacillus velezensis YtnP-homologous lactonase, gene and application thereof
  • Bacillus velezensis YtnP-homologous lactonase, gene and application thereof
  • Bacillus velezensis YtnP-homologous lactonase, gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Cloning of embodiment 1 Veles bacillus YtnP-homologous lactonase gene

[0081] (1) Extraction of Bacillus Velez genome DNA

[0082] The culture of Bacillus Velez was incubated at 37°C, shaken at 180rpm for 12 hours, and centrifuged at 8000rpm for 10min to obtain bacterial pellets. 100ng of genomic DNA of DH82 strain was extracted from the particles by CTAB method.

[0083] (2) Cloning of Bacillus Velez YtnP-homologous lactonase gene

[0084] Amplification of YtnP from Genomic DNA by PCR DH82 the sequence of

[0085] PCR primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0086] The primer sequences are:

[0087] Forward primer YtnP-F: 5′-GGAATTCATGGAGACATTGAATATTGGGAATTTTC-3′;

[0088] Reverse primer YtnP-R: 5'-GGAATTCATGGAGACATTGAATATTGGGAATATTTC-3'.

[0089] According to the operating instructions of the Takara PrimeSTAR kit, Bacillus beilesi DH82 genomic DNA (100ng) was used as a template.

[0090] PCR amplification program: pre-denaturati...

Embodiment 2

[0094] The construction of embodiment 2 Bacillus velei YtnP-homologous lactonase engineering bacteria

[0095] The PCR products were digested with restriction endonucleases NdeI and XhoI, respectively, and then ligated between multiple cloning sites on the pET28a vector with Takara ligation kit (T4 ligase). Expression cloning is driven by T7 promoter respectively, and the His tag coding sequence on the plasmid encodes histidine to the N-terminus of the target protein. Positive clones were amplified in Escherichia coli DH5α and transferred to Escherichia coli BL21 for protein expression.

Embodiment 3

[0096] The preparation of embodiment 3 Veles bacillus YtnP-homologous lactonase

[0097] Inoculation of 0.4 mM IPTG into bacterial cultures after 3 h induces YtnP DH82 The expression of Bacteria was harvested after 20 hours of cultivation, and the lysis buffer [300mM NaCl, 50mM NaH 2 PO4 (pH 7.4)] resuspended, then eluted with imidazole buffer [300mM NaCl, 200mM imidazole, 50mM NaH 2 PO4 (pH 7.4)] for washing. The target protein was purified by high-affinity NI-NTA chromatography. The purified protein was further analyzed by SDS-PAGE.

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Abstract

The invention discloses a bacillus velezensis YtnP-homologous lactonase, a gene and application thereof. The invention firstly discloses the bacillus velezensis YtnP-homologous lactonase, wherein the amino acid sequence of the bacillus velezensis YtnP-homologous lactonase is shown as SEQ ID NO.1. The invention further discloses the application of the bacillus velezensis YtnP-homologous lactonase in the treatment of dental chair type equipment. The disclosed bacillus velezensis YtnP-homologous lactonase can be used for remarkably inhibiting early proliferation, biofilm formation and virulence factor release of pseudomonas aeruginosa, is an effective way for reducing opportunistic pathogenic bacterium infection, and can be used as a novel dental hygiene treatment disinfectant.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a YtnP-homologous lactonase from bacillus velei and its gene and application. Background technique [0002] The water used by the modern dental chair unit (DCU) is supplied through the plumbing system of the dental unit water line (DUWL), which cools the integrated instruments and is used for tooth rinsing during dental treatment. Studies have shown that the number of bacteria (mainly Pseudomonas aeruginosa) in DCU water samples is much higher than the number of bacteria allowed in drinking water, and the bacteria adapt to the colonization of DUWL and the resistance to disinfectants through the formation of biofilm. Damage to health due to infection caused by force. Currently, standard water rinsing procedures cannot completely remove biofilms, the use of antibiotics can cause pathogenic bacteria to resist, and the use of chemicals such as glutaraldehyde is also harmful to pat...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/70C12N1/21A61K38/46A61P31/04A61P1/02C02F3/34C12R1/19
CPCC12N9/18C12N15/70A61P31/04A61P1/02C02F3/342C12Y301/01081A61K38/00Y02A50/30
Inventor 孙晓晖刘嘉周树锋
Owner HUAQIAO UNIVERSITY
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