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Staphylococcus lyase and application thereof

A staphylococcus and lyase technology, applied in the field of biological preparations, can solve the problems of narrow pH range, inability to adapt to high protein environment, insoluble expression and other problems

Active Publication Date: 2015-07-29
TARGET ANTI-BIOPHARMACEUTICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these lyases are difficult to express solublely, or have low activity, or cannot adapt to high-protein environments (such as milk, etc.), and the pH range of action is narrow, generally at pH 5-8

Method used

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  • Staphylococcus lyase and application thereof
  • Staphylococcus lyase and application thereof
  • Staphylococcus lyase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the construction of the lyase that can kill Staphylococcus.

[0028] (1) The DNA sequence of the ClyO gene capable of expressing the cleavage enzyme ClyO was synthesized in Shanghai Sangon Bioengineering Co., Ltd. in its entirety. The synthetic sequence was loaded into the pUC57 plasmid. Using the ClyO gene as a template, NcoI and XhoI restriction sites were added to both ends of the target gene, and the primers were designed as follows:

[0029] Forward primer: 5-TTAA CCATGG GCATGGCACTGCCTAAAACG-3 (SEQ.ID.NO.3)

[0030] NCOI

[0031] Reverse primer: 5-ATAT CTCGAG TTTAAATGTACCCCAAGC-3 (SEQ.ID.NO.4)

[0032] wxya

[0033] Using 2 μl of the gene as a template, add 1 μg of primers for PCR amplification. The PCR amplification procedure is as follows: 1) 94°C, 5min; 2) 94°C, 30sec, 62°C, 45sec, 72°C, 45sec, 30 cycles; 3 )72°C, 10min; the product was recovered by gel electrophoresis, and the electrophoresis pattern was as fol...

Embodiment 2

[0038] Embodiment 2, ClyO kills the verification of Staphylococcus aureus standard strain CCTCC AB91118

[0039] The overnight culture of Staphylococcus aureus was collected by centrifugation, washed once with phosphate buffered saline (PBS), and then resuspended in PBS solution. Take a certain amount of ClyO and mix it with the above-mentioned bacterial solution, and monitor the change of the absorption value of the mixed solution at 600nm with a microplate reader. A mixture of buffer and Staphylococcus aureus was used as a negative control. The final cracking curve obtained is shown in figure 2 . At the same time, the number of viable bacteria was calculated by diluting the plate after treatment for different times. The results show that ClyO can rapidly lyse the CCTCC AB91118 strain, resulting in a decrease in the turbidity of the bacterial liquid, which is manifested by a rapid decrease in the absorption value of the bacterial liquid at a wavelength of 600nm.

Embodiment 3

[0040] Example 3. Verification of ClyO's specificity to various staphylococci in vitro and to different strains.

[0041] In order to verify the bactericidal spectrum of ClyO, we selected several non-staphylococcus strains preserved in the inventor's laboratory and clinically isolated Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcus equineus, Staphylococcus squirrel, Hemolyticus Staphylococcus, Staphylococcus chromogenes, and other strains were tested together. Firstly, the tested strains were cultured overnight, collected by centrifugation, washed once with PBS, and then resuspended in PBS. A certain amount of ClyO was mixed with the above-mentioned bacterial solution respectively, and at the same time, the change of the absorption value of the mixed solution at 600 nm was monitored for 30 minutes with a microplate reader. use OD 600 The reduced rate indicates the lytic effect of the different strains. At the same time, the mi...

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Abstract

The invention discloses lyase capable of killing staphylococci and an application of the lyase, which belong to the field of biological agents. The invention discloses an amino acid sequence and a coding gene sequence of the lyase. A pH range of the lyase which plays a role is wide, the activity of cracking the staphylococci is kept within the pH range of 4 to 11, and recombinant protease constructed by adopting coding genes can be solubly expressed in colibacillus BL21(DE3). The lyase can be used for efficiently killing various staphylococci in vitro, including methicillin sensitive staphylococcus aureus and methicillin resistant staphylococcus aureus which are clinically separated, and the lyase can be used as antibiotics for treating staphylococcal infection in vivo. The disclosed lyase can be used for quickly cracking staphylococcal cell walls and releasing substances in cells, such as triphosadenine, DNA and the like, and the staphylococci can be detected by utilizing the released substances.

Description

technical field [0001] The invention belongs to the field of biological preparations, and in particular relates to a lyase capable of killing staphylococcus, especially staphylococcus aureus and its application in aspects such as killing and detecting staphylococci. Background technique [0002] Staphylococcus can be divided into three types according to biochemical reactions and pigment production: Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophytics. Among them, Staphylococcus aureus is mostly pathogenic bacteria, Staphylococcus epidermidis is occasionally pathogenic, and Staphylococcus saprophyticus is generally not pathogenic. Staphylococcus aureus (staphylococcus aureus) is a common Gram-positive coccus that can cause many serious infections in humans and animals, and is also one of the common pathogens of hospital infection. Staphylococci are susceptible to resistance to antibiotics, including the usual variety, as well as newer antimicrob...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12Q1/68C12Q1/66C12Q1/527C12Q1/14A61K38/51A61P31/04
CPCA61K38/51C12N9/88C12Q1/14C12Q1/527C12Q1/66C12Q1/689A61K38/00C12Q1/68C12N15/70C12N1/06C12N9/485C12N9/80C12N15/62C12N9/2462A61P31/04C12Q1/6806
Inventor 危宏平杨航余军平
Owner TARGET ANTI-BIOPHARMACEUTICAL TECH CO LTD
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