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Klebsiella pneumoniae phage P560, phage depolymerizing enzyme Depo43 and application

A technology of Klebsiella and phage, applied in the direction of phage, virus/phage, application, etc., can solve the problem that the actual action spectrum of phage depolymerase is not disclosed, and achieve significant clinical effects and efficient inhibitory effects

Active Publication Date: 2021-03-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patent with the application number CN201811349779.2 and titled "Phage depolymerase for degrading Klebsiella pneumoniae capsular polysaccharide and biofilm" discloses a phage depolymerase and its application, but does not disclose the phage depolymerase. The actual spectrum of action of the polymerase, and there is no indication of any application prospects for this phage depolymerase

Method used

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  • Klebsiella pneumoniae phage P560, phage depolymerizing enzyme Depo43 and application
  • Klebsiella pneumoniae phage P560, phage depolymerizing enzyme Depo43 and application
  • Klebsiella pneumoniae phage P560, phage depolymerizing enzyme Depo43 and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Preparation of phage depolymerase Depo43

[0027] (1) Target product amplification

[0028] Primers designed for constructing depolymerase expression vector:

[0029] The upstream primer F (SEQ ID No: 3) is:

[0030] 5'-GGAATTCCATATGTTAAAACAATCTGAATCAG-3';

[0031] The downstream primer R (SEQ ID No: 4) is:

[0032] 5'-CCGCTCGAGTTATGGACCAATGACCACACC-3'.

[0033] The PCR amplification program was denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 50°C for 15 s, extension at 72°C for 2 min, and 30 cycles.

[0034] The depo43 gene PCR product from the 43rd open reading frame of Klebsiella pneumoniae phage P560 was digested and purified and ligated into the pET28a plasmid: restriction sites Nde I and Xho I;

[0035] (2) Add the pET28a-depo43 plasmid to 100 μL Escherichia coli BL21 (DE3) competent cells, flick the tube wall to mix, and let stand on ice for 30 minutes. After heat-shocking in a water bath at 42°C for 45 sec, quickly pla...

Embodiment 2

[0046] Example 2 Using phage depolymerase Depo43 to remove K47 and K64 type bacterial capsular polysaccharides respectively

[0047] Use the dot test to detect the inhibitory activity of phage depolymerase Depo43 on E. coli biofilm. Spread 0.5% semi-solid LB medium containing 100 μL of logarithmic bacteria evenly on the solid LB medium, and dry it naturally. A series of diluted concentrations of Dpo43 (2 mg / mL, 0.2 mg / mL, 0.02 mg / mL, 2 μg / mL, 0.2 μg / mL, 0.02 μg / mL, 2 ng / mL and 0.2 ng / mL) were dropped on the double layer plate , the buffer was used as a negative control, cultured overnight in a 37°C incubator, and the phage depolymerase Depo43 with a concentration of 2 mg / mL-0.2 μg / mL formed translucent spots on the plate, such as image 3 shown.

[0048] The extracted capsular polysaccharide was added to a 10% gel (final concentration: 0.3%). After solidification, a 7 mm hole was punched into the gel, and then 20 μL of purified protein and buffer (negative control) were added...

Embodiment 3

[0049] Example 3 Utilizes purified depolymerase depo43 to inhibit K47 type Klebsiella pneumoniae biofilm

[0050] K47 Klebsiella pneumoniae Kp57 80μL (OD 0.4~2×10 8 CFU / mL) were inoculated into a 96-well cell culture plate (Sigma-Aldrich, USA) containing 100 μL LB medium per well. Dilute the purified depo43 with protein buffer to 200 μg / mL, add 20 μL depo43 dilution to each well of the 96-well cell culture plate containing the biofilm, the content is 4 μg, 0.4 μg, 0.04 μg, 4ng, 0.4ng, 0.04 ng and 0.004ng. After culturing for 24 hours, the cell culture plate was taken out, the medium was discarded with a pipette gun and washed twice with sterilized PBS buffer to remove free bacteria and enzymes. Then add 200 μL methanol to each well and fix for 10 minutes. After the fixative was discarded and allowed to dry naturally, the biofilm was stained with 200 μL of 0.1% crystal violet (purchased from Zhuhai Beisuo Biotechnology Co., Ltd.) for 10 minutes at room temperature. After st...

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Abstract

The invention discloses a phage depolymerizing enzyme Depo43 based on a klebsiella pneumoniae phage P560 and an application of the phage depolymerizing enzyme Depo43. The phage P560 disclosed by the invention is preserved in China Center for Type Culture Collection on October 26, 2020, the preservation name is Klebsiella pneumonia phage P560, and the preservation number is CCTCC NO: M 2020643. A 43rd open reading frame of the phage P560 is subjected to prokaryotic expression and purification to obtain the phage depolymerizing enzyme Depo43, and the depolymerizing enzyme not only can remove capsular polysaccharides formed by K47 and K64 klebsiella pneumoniae, but also has an efficient inhibition effect on the formation of biofilms. The epolymerizing enzyme can be widely applied to preparation of antibiotic substitute preparations and medical instrument disinfectants, and has a remarkable clinical effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Klebsiella pneumoniae phage strain P560, a depolymerase Depo43 derived from the Klebsiella pneumoniae phage strain P560, and preparation and application of the depolymerase Depo43. Background technique [0002] Klebsiella pneumoniae is a common zoonotic pathogen. In recent years, multidrug-resistant Klebsiella pneumoniae has become one of the most important pathogens of hospital-acquired infections. The bacterium not only poses a huge threat to human public health, but also affects the healthy development of the animal breeding industry to a certain extent. [0003] Most Klebsiella pneumoniae have the ability to synthesize and secrete capsular polysaccharides. Capsular polysaccharides serve as a natural barrier for bacteria to maintain bacterial virulence, adherence, and block penetration of some antibiotics. As an important virulence factor of Klebsiella pneumoniae,...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N9/24C12N15/56C12N15/70A01N47/44A01P1/00C12R1/92
CPCC12N7/00C12N9/2402C12N15/70A01N47/44C12N2795/00021
Inventor 张炜李敏杜鸿李培肖宇屹
Owner NANJING AGRICULTURAL UNIVERSITY
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