Streptococcus broad-spectrum chimeric lyase GBS-V12b and coding gene and application thereof

A streptococcus and lyase technology, applied in the field of genetic engineering, can solve problems such as mixed bacterial infection and narrow lysis spectrum, and achieve high stability and wide application prospects

Active Publication Date: 2017-08-08
武汉中科瑞昊科技发展有限公司
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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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  • Streptococcus broad-spectrum chimeric lyase GBS-V12b and coding gene and application thereof
  • Streptococcus broad-spectrum chimeric lyase GBS-V12b and coding gene and application thereof
  • Streptococcus broad-spectrum chimeric lyase GBS-V12b and coding 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 PlyV12

[0029] The full-sequence synthesis of lyase PlyGBS and lyase PlyV12 (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 PlyV12 gene as a template and V12b-F / V12b-R as primers, the cell wall binding of PlyV12 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] V12b-F:5-ATAT GGATCC TTAAACGGTGGAAGCAC-3

[0033] V12b-R:5-TATA CTCGAG CTTAAATGTACCCCATG-3

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

Embodiment 2

[0041] Example 2: Verification of GBS-V12b 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-V12b in Example 1 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 OD after mixing Streptococcus dysgalactiae and GBS-V12b 600 The trend of change over time, the results show that GBS-V12b can rapidly lyse the ATCC 35666 strain, resulting ...

Embodiment 3

[0043] Example 3, GBS-V12b 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 0.01mL of GBS-V12b and 0.19mL of the above-mentioned bacterial solution, mix and incubate at 37°C for 60min, and monitor the change of the absorption value of the mixture at 600nm with a microplate reader. The lysis effect of different strains is indicated by the value of OD600 reduction. At the same time, the mixed solution of 0.01mL PBS buffer solution and 0.19mL bacterial solution was used as a negative control, and the killing effect obtained by the test was as follows: image 3 shown. The results show that GBS-V12b can quickly kill a variety of streptococci (including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus suis, Streptococc...

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Abstract

The invention relates to the technical field of genetic engineering and particularly relates to a streptococcus broad-spectrum chimeric lyase GBS-V12b and a coding gene and application thereof. The streptococcus broad-spectrum chimeric lyase GBS-V12b has an amino acid sequence shown in SEQ ID No. 1. The coding gene of the streptococcus broad-spectrum chimeric lyase GBS-V12b has a nucleotide sequence shown in SEQ ID No. 2. The streptococcus broad-spectrum chimeric lyase GBS-V12b disclosed by the invention can be used for killing a variety of streptococci and staphylococci, particularly streptococcus pneumoniae, streptococcus pyogenes, streptococcus suis, streptococcus agalactiae, streptococcus dysgalactiae, streptococcus mutans, streptococcus iniae, and a variety of enterococci and staphylococcus aureus; the GBS-V12b is good in stability and is insensitive to high-concentration NaCl; the recombinant protein GBS-V12b can be excellently expressed in Escherichia coli BL21 (DE3); high-dosage GBS-V12b is free of obvious cytotoxicity. Therefore, the GBS-V12b can be used for controlling a variety of streptococci and treating infections caused by the streptococci independently or as an additive, thereby having a broad application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a streptococcus broad-spectrum chimeric lyase GBS-V12b and its coding gene and application. Background technique [0002] Streptococci are spherical, Gram-positive bacteria that cause many diseases in humans and animals. There are many different types of strep, and different types of strep can cause different types of infections. Several major streptococcal pathogens include Streptococcus pneumoniae, Group B Streptococcus, Streptococcus suis, Streptococcus mutans, and Streptococcus pyogenes. Among them, Streptococcus pneumoniae is one of the main pathogens causing lobar pneumonia, which is very common in infants and young children with bronchopneumonia. Group B Streptococcus is one of the important pathogens of perinatal infection, which seriously threatens the health of newborns. Moreover, group B streptococci can also cause fish streptococcosis in the...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60A61K38/51A61P31/04A61P1/02
CPCA61K38/00C12N9/88
Inventor 危宏平杨航洪伟
Owner 武汉中科瑞昊科技发展有限公司
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