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Novel broad-spectrum chimeric lysin BGS-PlySb and encoding gene and application thereof

A lyase, a new type of technology, applied in the field of genetic engineering, can solve the problems of narrow lysis spectrum, can not meet the mixed infection of bacteria, etc., achieve the effect of high stability and wide application prospects

Active Publication Date: 2017-09-19
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the strong host selectivity of the lyase, it usually has a narrow cleavage spectrum, which cannot meet the needs of bacterial mixed infection

Method used

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  • Novel broad-spectrum chimeric lysin BGS-PlySb and encoding gene and application thereof
  • Novel broad-spectrum chimeric lysin BGS-PlySb and encoding gene and application thereof
  • Novel broad-spectrum chimeric lysin BGS-PlySb and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1: Construction of broad-spectrum streptococcus chimeric lyase

[0028] 1. Preparation of the catalytic domain of PlyGBS and the cell wall binding domain of PlySs2

[0029] The full-sequence synthesis of lyase PlyGBS and lyase PlySs2 (Nanjing GenScript Biotechnology Co., Ltd.), and the synthetic sequence was loaded into the pUC57 plasmid. Using the plyGBS gene as a template and GBS180-F / GBS180-R as primers, the sequence of the catalytic domain GBS180 of PlyGBS was amplified. Using the plySs2 gene as a template and PlySb-F / PlySb-R as primers, the cell wall binding of PlySs2 was amplified. area. The primers and restriction site information used in the clone construction are as follows:

[0030] GBS180-F:5-TTAA CCATGG GCATGGCTACCTACCAGG-3

[0031] GBS180-R:5-TATA GGATCC GATCGTTTTGGTCGTGC-3

[0032] PlySb-F:5-TATA GGATCC TCTCGTTCCTATCGCGAG-3

[0033] PlySb-R:5-TATA CTCGAG TTTAAATGTACCCCCAAG-3

[0034] GBS180-F primers underline the restriction endon...

Embodiment 2

[0041] Example 2: Verification of GBS-PlySb killing Streptococcus dysgalactiae standard strain ATCC 35666

[0042] Cultivate Streptococcus dysgalactiae overnight, collect by centrifugation, wash once with PBS, then dissolve in PBS, and adjust the OD of the bacteria solution 600nm =0.6~0.8. Take 0.01mL of GBS-PlySb enzyme solution and mix it with 0.19mL of the above bacterial solution, and monitor the change of the absorption value of the mixture at 600nm with a microplate reader. At the same time, a mixture of 0.01 mL of PBS buffer solution and 0.19 mL of Streptococcus dysgalactiae was used as a negative control. The final cracking curve obtained is figure 2 As shown, the two curves respectively represent the OD after Streptococcus dysgalactiae was mixed with the buffer 600 The trend of change over time and the OD of Streptococcus dysgalactiae mixed with GBS-PlySb 600 The trend of change over time, the results show that GBS-PlySb can rapidly lyse the ATCC 35666 strain, re...

Embodiment 3

[0043] Example 3, GBS-PlySb broad-spectrum verification of killing streptococci in vitro

[0044] Various strains of Streptococcus, Staphylococcus aureus, Enterococcus and Escherichia coli were cultured overnight, collected by centrifugation, washed once with PBS and then dissolved in PBS, and adjusted to OD600nm=0.6-0.8. Take 0.01 mL of GBS-PlySb enzyme solution and mix it with the above bacterial solution and incubate at 37°C for 60 min, while monitoring the change of the absorption value of the mixture at 600 nm with a microplate reader. The lysis effect of different strains is indicated by the value of OD600 reduction. At the same time, the mixture of 0.01mL PBS buffer solution and 0.19mL bacterial solution was used as a negative control, and the killing effect obtained in the test was shown in image 3 shown. The results show that GBS-PlySb enzyme can quickly kill a variety of streptococci (including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, ...

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Abstract

The invention relates to the technical field of gene engineering, in particular to novel broad-spectrum chimeric lysin BGS-PlySb and an encoding gene and application thereof. The novel broad-spectrum chimeric lysin BGS-PlySb has an amino acid sequence shown as in SEQ ID NO. 1; the encoding gene of the novel broad-spectrum chimeric lysin BGS-PlySb has a nucleotide sequence shown as in SEQ ID NO. 2. A novel chimeric lysin is constructed by means of gene splicing and is suitable for killing various species of Streptococcus and Staphylococcus, particularly various species of Enterococcus, and Staphylococcus aureus, as well as Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus mutans, Streptococcus iniae and other species; the novel broad-spectrum chimeric lysin BGS-PlySb has good stability and is insensitive to high-concentration NaCl, recombinant protein GBS-PlySb is well expressible in Escherichia coli BL21 (DE3), and a high dose of GBS-PlySb is free of significant cytotoxicity. Therefore, the novel broad-spectrum chimeric lysin BGS-PlySb is applicable independently to or as an additive to the control of various species of Streptococcus and the treatment of infections caused by these species, and has a promising application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a novel broad-spectrum chimeric lyase GBS-PlySb and its coding gene and application. Background technique [0002] Streptococci are spherical, Gram-positive bacteria that cause a variety of diseases in humans and animals. Several major streptococcal pathogens include Streptococcus pneumoniae, Group B Streptococcus, Streptococcus suis, Streptococcus mutans, and Streptococcus pyogenes. Streptococcus pneumoniae is one of the main pathogenic bacteria causing nosocomial infections. Group B Streptococcus, especially Streptococcus agalactiae, can cause a variety of infections in humans and animals. In the hospital, Streptococcus agalactiae can cause perinatal infection of pregnant women, which seriously threatens the health of newborns; serious. Streptococcus suis is a zoonotic acute and febrile infectious disease, once infected, it will cause great harm. St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60A61K38/51A61P31/04A61P1/02
CPCA61K38/00C12N9/88
Inventor 危宏平杨航洪伟卢刚
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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