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A vibrio parahaemolyticus lyase, its coding gene and its application

A technology of Vibrio hemolyticus and encoding gene, applied in the directions of lyase, application, genetic engineering, etc., can solve problems such as reducing effectiveness, and achieve the effects of high specificity, wide host spectrum, and high lysis efficiency

Active Publication Date: 2022-03-08
湖北小胡鸭酱卤食品研究院有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, phages are highly immunogenic drugs, which can reduce their effectiveness for repeated use

Method used

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  • A vibrio parahaemolyticus lyase, its coding gene and its application
  • A vibrio parahaemolyticus lyase, its coding gene and its application
  • A vibrio parahaemolyticus lyase, its coding gene and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The preparation of embodiment 1 Vibrio parahaemolyticus lyase

[0022] 1.1 Isolation, purification and morphological observation of Vibrio parahaemolyticus phage F23s1

[0023] The samples taken from the seafood market were cultured overnight with Vibrio parahaemolyticus F23 cultured to the logarithmic phase for 12h to 18h, and filtered through a 0.22μm microporous membrane to obtain the phage stock solution. Use the double-layer plate method to purify the phage, and continue to purify until the size and transparency of the phage plaques appearing on the double-layer plate are consistent, which is the purified phage, named Vibrio parahaemolyticus phage F23s1. After the Vibrio parahaemolyticus phage F23s1 was negatively stained with phosphotungstic acid, it was placed under a projection electron microscope to observe the phage morphology. The specific operation steps were as follows: after immersing the copper grid in the phage liquid for 10 minutes, absorb the excess li...

Embodiment 2

[0059] The antibacterial effect of embodiment 2 Vibrio parahaemolyticus lyase to Vibrio parahaemolyticus F23

[0060] Using the turbidity method to detect, inoculate the host bacteria Vibrio parahaemolyticus F23 to grow overnight in LB liquid medium, transfer to 25mL medium at a ratio of 1:100 and shake for 3 hours, add chloroform to a final concentration of 0.5% (v / v) , let stand for 15 minutes after gentle shaking, centrifuge and wash 3 times with sterile deionized water, resuspend the pellet in 50mM Tris-HCl (pH 8.2) buffer containing 0.1% Triton X-100, and adjust the OD600nm to 0.8-1.0 , add 50 μL of lyase (final concentration of 20, 10, 5, 2, 1, 0.5, 0.1 μM) to 200 μL of bacterial suspension, use the same volume of TrisHCl buffer containing 0.1% Triton X-100 instead of lysis The enzyme was used as a control, and its OD value was measured at 600nm. The result is as Figure 4 As shown, different concentrations of lyase can effectively lyse Vibrio parahaemolyticus F23.

Embodiment 3

[0061] The host spectrum of embodiment 3 Vibrio parahaemolyticus lyase

[0062] The 23 kinds of Vibrio parahaemolyticus, Escherichia coli and Salmonella in Example 1 were respectively verified for the host spectrum of Vibrio parahaemolyticus lyase, and different bacteria were inoculated to grow overnight in LB liquid medium, and then transferred at a ratio of 1:100 to Shake culture in 25mL culture medium for 3h, add chloroform to a final concentration of 0.5% (v / v), let it stand for 15min after gentle shaking, centrifuge and wash 3 times with sterile deionized water, and resuspend the precipitate in a solution containing 0.1% Triton X- 100 in 50mM Tris-HCl (pH 8.2) buffer, and adjust OD600nm to 0.8-1.0, add 50μL lyase (final concentration 0.1μM) to 200μL bacterial suspension, use the same volume of 0.1% Triton The Tris-HCl buffer solution of X-100 replaces the lyase as a control, observes and measures the OD600nm value when acting for 30 minutes, and the results are as follows...

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Abstract

The invention relates to a Vibrio parahaemolyticus lyase, the amino acid sequence of which is shown in SEQ ID NO:1. The present invention also relates to a Vibrio parahaemolyticus lyase encoding gene, which encodes a protein having the sequence shown in SEQ ID NO:1. The present invention also provides the application of the above Vibrio parahaemolyticus lyase in lysing Vibrio parahaemolyticus. The vibrio parahaemolyticus lyase can lyse the vibrio parahaemolyticus, has high specificity for the vibrio parahaemolyticus, high lysis efficiency, and the host spectrum of the vibrio parahaemolyticus is wide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Vibrio parahaemolyticus lyase, its coding gene and its application in lysing Vibrio parahaemolyticus. Background technique [0002] Vibrio parahaemolyticus (V.parahaemolyticus) is a halophilic Gram-negative bacterium, which is an important food-borne pathogen worldwide, mainly distributed in the marine environment. Consumption of seafood contaminated by Vibrio parahaemolyticus can cause gastrointestinal disturbances resulting in symptoms of diarrhea, nausea and, in severe cases, death. Vibrio parahaemolyticus has become a biohazard in aquaculture, and humans use antibiotics to prevent contamination. Antibiotics work again as antibiotic use leads to the rapid spread of multi-antibiotic resistance in bacteria. With the emergence of extensively drug-resistant bacterial pathogens, phage therapy and other alternative or additional treatment modalities have received renewed attention. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A01N63/50A01P1/00C12R1/19
CPCC12N9/88C12N15/70A01N63/50A01N63/40
Inventor 周敏杨后继夏海王宏勋侯温甫
Owner 湖北小胡鸭酱卤食品研究院有限公司
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