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High-efficiency streptococcus pneumonia chimeric lyase and its mutant and application thereof

A Streptococcus pneumoniae, lysing enzyme technology, applied in the field of genetic engineering, can solve the problems that the physical and chemical properties cannot meet the requirements of Streptococcus pneumoniae infection, the number is small, and achieve the prevention and treatment of Streptococcus pneumoniae infection, good stability, and efficient killing Effect

Active Publication Date: 2018-11-16
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

[0005] At present, some lyases of Streptococcus pneumoniae have been reported. They have different characteristics in bactericidal activity and stability, but the number is still relatively small, and some physical and chemical properties cannot meet the actual needs of controlling Streptococcus pneumoniae infection.

Method used

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  • High-efficiency streptococcus pneumonia chimeric lyase and its mutant and application thereof
  • High-efficiency streptococcus pneumonia chimeric lyase and its mutant and application thereof
  • High-efficiency streptococcus pneumonia chimeric lyase and its mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Synthesis of the cell wall binding domain GPB of ACQ35_gp20

[0043] The ACQ35-gp20 gene of Streptococcus phage SpSL1 was synthesized in Nanjing GenScript Biotechnology Co., Ltd., and the synthetic sequence was loaded into pUC57 plasmid. Using the ACQ35-gp20 gene as a template and gp20-F / gp20-R as primers, the cell wall binding domain GPB of ACQ35-gp20 was amplified, its amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO. 4. The primers and restriction site information used in the clone construction are as follows:

[0044] SEQ ID NO.11 gp20-F:5-tataggatccgttgatccgtatccgtatctg-3BamHI

[0045] SEQ ID NO.12 gp20-R:5-tatactcgagtttggtggtaatcagaccgtcc-3XhoI

[0046] The reaction system for PCR amplification of gene fragments is:

[0047]

[0048] The PCR amplification procedure is as follows:

[0049] 1) 94°C, 5min; 2) 94°C, 30sec, 62°C, 45sec, 72°C, 45sec, 30 cycles; 3) 72°C, 10min.

Embodiment 2

[0050] Example 2: Construction and expression of EGFP-GPB and EGFP

[0051] 1. The ACQ35-gp20 gene of Streptococcus phage SpSL1 was synthesized in Nanjing GenScript Biotechnology Co., Ltd., and the synthetic sequence was loaded into the pUC57 plasmid. Using the pEGFP plasmid as a template and EGFP-F / EGFP-R as primers, the sequence of enhanced green fluorescent protein EGFP was amplified. The primers and restriction site information used in the construction of the clone are as follows:

[0052] SEQ ID NO.13 EGFP-F:5-tataccatggtgagcaagggcgaggagc-3NcoI

[0053] SEQ ID NO.14 EGFP-R:5-tataggatcccttgtacagctcgtccatgc-3BamHI

[0054] The reaction system for PCR amplification of gene fragments is:

[0055]

[0056]

[0057] The PCR amplification procedure is as follows:

[0058] 1) 94°C, 5min; 2) 94°C, 30sec, 62°C, 45sec, 72°C, 45sec, 30 cycles; 3) 72°C, 10min.

[0059] 2. The above fragment and the cell wall binding domain GPB of ACQ35-gp20 in Example 1 were digested with co...

Embodiment 3

[0061] Example 3: Validation of EGFP-GPB binding to Streptococcus pneumoniae

[0062] Overnight cultures of streptococci including Streptococcus pneumoniae NS26 (Spn NS26), Streptococcus pneumoniae NS63 (SpnNS63), Streptococcus agalactiae (Sag), Streptococcus dysgalactiae (Sdy), Streptococcus pyogenes (Spy), and Streptococcus mutans For cocci (Smu), wash once with PBS and resuspend to OD600=0.5-0.8, then take 0.01 ml of the treated bacteria solution and incubate with 20 μM EGFP-GPB protein at 37 degrees for 1 hour. Wash 3 times with PBS to remove unbound protein, and observe the cells under a fluorescent microscope. At the same time, under the same experimental conditions, EGFP protein was used as a control protein for binding experiments. The result obtained is as figure 2 shown. The results showed that EGFP-GPB could specifically bind to Streptococcus pneumoniae strains.

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Abstract

The invention relates to the technical field of gene engineering, and especially relates to high-efficiency streptococcus pneumonia chimeric lyase and its mutant and an application thereof. A gene splicing means is employed, a brand new chimeric lyase ClyJ is constructed, and the mutants ClyJ-1, ClyJ-2 and ClyJ-3 are constructed through gene fragment insertion and deletion. The ClyJ and its mutanthave good stability, and efficiently kill streptococcus pneumonia both in vitro and in vivo; the ClyJ and its mutant can better expressed in Escherichia coli BL21(DE3), and have latent prevention andtreatment capability for streptococcus pneumonia infection through high-efficiency cracking activity on streptococcus pneumonia. the ClyJ and its mutant can be individually used, or can be used as anadditive for compatibility with different forms of reagents and solutions, and have large application prospect for controlling the streptococcus pneumonia and treating infection due to streptococcuspneumonia.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a high-efficiency Streptococcus pneumoniae chimeric lyase and its mutant and application. Background technique [0002] Streptococcus pneumoniae (Streptococcus pneumoniae) is a class of capsulated Gram-positive bacteria, which is an opportunistic pathogenic bacteria and is widely distributed in the nasopharynx of humans and animals. Infection with Streptococcus pneumoniae causes a range of invasive diseases, the most common of which are acquired pneumonia, bacteremia, and meningitis. In addition, Streptococcus pneumoniae can also cause other mucosal infection diseases due to airborne transmission, such as acute otitis media, sinusitis and bronchitis. Due to the overuse of antibiotics, multidrug-resistant Streptococcus pneumoniae isolates continue to appear worldwide, posing severe challenges to clinical treatment. In addition, there are as many as 92 serotypes of St...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70A61K38/51A61P31/04
CPCA61K38/00A61P31/04C12N9/88C12N15/70
Inventor 危宏平杨航
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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