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Recombinant bacillus subtilis and application thereof

A technology of Bacillus subtilis and bacitracin, applied in the field of construction of Bacillus subtilis engineering bacteria, can solve the problems of few reports on biodegradable polypeptide antibiotics

Active Publication Date: 2018-12-21
浙江科峰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the literature analysis of the past 50 years at home and abroad shows that the current research work on polypeptide antibiotics at home and abroad is mainly in the aspects of pharmacokinetics, pharmacodynamic analysis, drug purification and drug resistance detection. Research has rarely reported

Method used

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  • Recombinant bacillus subtilis and application thereof
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  • Recombinant bacillus subtilis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Construction of recombinant shuttle plasmid pWGPapr

[0028] 1. PCR amplification of the polypeptide antibiotic degrading enzyme gene fragment papr.

[0029] Using the genome of Bacillus licheniformis ATCC 14580 as a template, using PaprF and PaprR containing restriction enzyme sites as primers, PCR amplifies the target fragment papr of the polypeptide antibiotic degrading enzyme gene, and the amplified product is detected by 1% agarose gel electrophoresis , the size of the PCR product is 1539bp (the size is 1539bp, including the promoter part, the sequence is shown in SEQ ID NO.1). After purification, restriction endonucleases Xho I and BamH I (Dalian TaKaRa Company) were used for double digestion, purified and recovered for use.

[0030] Upstream primer (PaprF: 5'-CCG CTCGAG ATCTTTCACCCGTTTCTG-3', the underline is the Xho I restriction site);

[0031] Downstream primer (PaprR: 5'-CGC GGATCC TTATGGAGCGGCAGCTTC-3', the underline is the BamH I restric...

Embodiment 2

[0042] Example 2: Construction of recombinant Bacillus subtilis containing recombinant shuttle plasmid pWGPapr.

[0043] Utilize the plasmid extraction kit (Sangon Bioengineering (Shanghai) Co., Ltd.) to extract the recombinant shuttle plasmid pWGPapr from the Escherichia coli DH5α positive transformant containing the recombinant shuttle plasmid pWGPapr constructed in Example 1, and adopt the electroporation method in Under the conditions of voltage 21kV / cm and time constant = 5ms, the recombinant shuttle vector pWGPapr was transformed into Bacillus subtilis WB800 strain, screened on a 50μg / mL kanamycin-resistant LB plate, and obtained after colony PCR and sequencing verification. Bacillus subtilis WB800 / pWBUC01-apr of the recombinant shuttle plasmid pWGPapr, which is Bacillus subtilis WB800dc that degrades polypeptide antibiotics, was deposited in the China Center for Type Culture Collection on November 27, 2017, with a deposit number of It is: CCTCCNO:M 2017732, and the pres...

Embodiment 3

[0045] Example 3: Preliminary study on the degradation ability of Bacillus subtilis WB800dc to polymyxin E, a polypeptide antibiotic.

[0046] 1. Preparation of seed suspension:

[0047] LB liquid medium: Yeast powder 5.0g / L, tryptone 10.0g / L, sodium chloride 10.0g / L, solvent is deionized water, pH 7.0~7.2, filled with 50mL / 250mL triangular flask, sterilized at 121℃ 20min.

[0048] LB solid medium: yeast powder 5.0g / L, tryptone 10.0g / L, sodium chloride 10.0g / L, agar 20.0g / L, solvent is deionized water, pH 7.0~7.2, liquid 100mL / 250mL triangle Bottles, sterilized at 121°C for 20 minutes.

[0049] Streak inoculation of Bacillus subtilis WB800dc cells on LB solid medium containing 50 μg / mL kanamycin for activation, after inverting at 37°C for 24 hours, pick a single colony and inoculate in LB liquid medium, at 37°C, 200r Cultivate for 24 hours / min for further activation; then inoculate into LB liquid medium according to the volume concentration of 8% for expanded culture for 48...

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Abstract

The invention discloses recombinant bacillus subtilis and application thereof in degradation of polypeptide antibiotics. The engineering bacteria are obtained by ligating a gene segment shown in SEQ ID No. 1 to a linearized pWBUC01 vector and then transferring into bacillus subtilis. The bacillus subtilis engineering bacteria for degrading the polypeptide antibiotics can effectively and quickly remove the polypeptide antibiotics in a water environment, and has the advantages of safety, high efficiency and energy conservation compared with the traditional chemical degradation and physical absorption. The bacillus subtilis WB800 / pWBUC01-apr provided by the invention can effectively degrade polymyxin residue in the contaminated sludge, and the 16000 U / mL polymyxin is basically degraded completely in 8 days; however, the polymyxin degradation rate of WB800 with no-loaded pWBUC01 is only about 38%.

Description

(1) Technical field [0001] The invention relates to the construction and application of a Bacillus subtilis engineering bacterium used for the degradation of polypeptide antibiotics. (2) Background technology [0002] Antibiotics are chemical substances that can interfere or inhibit the physiological functions of other organisms at low concentrations. Antibiotics commonly used in clinical practice include extracts from microbial culture fluid and chemically synthesized or semi-synthesized compounds. Its discovery and application have played a huge role in human medical health and animal and plant disease prevention and control. However, due to the short retention time of antibiotics in organisms and low metabolic efficiency, most antibiotics are discharged into the environment in the form of original drugs or metabolites with feces, causing serious environmental pollution and endangering human health. Polypeptide antibiotics are a class of antibiotics with polypeptide stru...

Claims

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Application Information

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IPC IPC(8): C12N1/21C02F3/34C12R1/125C02F101/38
CPCC02F3/34C02F2101/38C12N9/54
Inventor 余志良音建华裘娟萍孙东昌赵春田王刚
Owner 浙江科峰生物技术有限公司
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