Lyase plyssX609 for lysing Gram-positive bacteria and application thereof

A Gram-positive bacteria and lyase technology, applied in the field of genetic engineering, can solve the problems of narrow cleavage spectrum, low cleavage efficiency, poor stability, etc.

Active Publication Date: 2021-05-07
武汉道前生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The clinical treatment of streptococcal infection mainly adopts traditional antibiotic therapy. In recent years, with the continuous emergence of the problem of poor antibiotic resistance, it is urgent to find a new and efficient antibiotic alternative therapy to deal with the increasingly serious problem of bacterial drug resistance. ; and the current lyase reported in the prior art still has the problems of narrow cleavage spectrum, low cleavage efficiency and poor stability

Method used

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  • Lyase plyssX609 for lysing Gram-positive bacteria and application thereof
  • Lyase plyssX609 for lysing Gram-positive bacteria and application thereof
  • Lyase plyssX609 for lysing Gram-positive bacteria and application thereof

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preparation example Construction

[0034] The present invention provides a method for preparing the above-mentioned recombinant bacteria, comprising the following steps:

[0035] The above-mentioned recombinant expression vector was used to transform the competent cells, and the plasmid was extracted and introduced into Escherichia coli to obtain recombinant Escherichia coli, namely, recombinant Escherichia coli BL21-pET28a-plyssX609. The Escherichia coli described in the present invention is preferably Escherichia coli BL21 (DE3); the transformation method is preferably chemical transformation.

[0036] The present invention provides the application of the above-mentioned recombinant expression vector or the above-mentioned recombinant bacterium or the recombinant bacterium prepared by the above-mentioned preparation method in the preparation of a reagent for lysing Gram-positive bacteria. The reagent provided by the invention has the advantages of good stability and wide cracking spectrum, and can efficiently...

Embodiment 1

[0045] Construction of Lyase plyssX609 Recombinant Expression Vector

[0046] (1) Download the streptococcus sequence from NCBI, the strain Genbank number is WP_161496994, the lyase fragment was synthesized by Wuhan Tianyi Huiyuan Company, and both ends have HindIIII and KpnI restriction sites.

[0047] (2) Carry out double enzyme digestion on the vector pet28a and the synthetic product in (1), the enzyme digestion sites are KpnI and HindIII, the system is 40 μl, wherein: the vector double enzyme digestion system: vector pET28a 7 μl, each of the two enzymes 1.5 μl, 10×buffer4μl, ddH 2 O26μl; recovered product double enzyme digestion system: step (1) recovered product 7μl, two enzymes each 1.5μl, 10×buffer4μl and ddH 2 O26μl; digestion at 37°C for 2h. After recovery, connect T4 overnight at 4°C, 20 μl of the system, including 40 ng of vector pET28a, 120 ng of recovered product in step (1), 1 μl of T4 enzyme; 2 μl of 10×T4 buffer, and ddH for the rest 2 0 to make up to 20 μl....

Embodiment 2

[0050] Expression and Purification of Lyase plyssX609 Protein

[0051] (1) The correctly sequenced pET28a-plyssX609 was chemically transformed into BL21 (DE3) competent cells. After expanding the culture, it was induced with IPTG at 16°C for 18 hours. After the bacterial solution was centrifuged, it was resuspended in PBS and crushed with an ultrasonic breaker. Ultrasonic 3s, intermittent 6s. After crushing, the supernatant was collected by centrifugation at 12,000 rpm, and filtered through a 0.22 μm filter to obtain unpurified lyase plyssX609 protein.

[0052] (2) Using Ni-His affinity purification, pass the unpurified protein through HiTrap Q Sepharose FF column (GEHealthy Care), and imidazole gradient elution, collect the effluent, and replace the effluent in PBS through a dialysis bag to remove residual imidazole. Finally, ultrafiltration was performed through a 3kDa ultrafiltration tube to obtain the concentrated purified protein.

[0053] The purified protein was iden...

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Abstract

The invention provides a lyase plssX609 for lysing Gram-positive bacteria and application thereof, and particularly relates to the field of genetic engineering. The invention provides a lyase glyssX609 for lysing Gram-positive bacteria, and the lyase glyssX609 is characterized in that the nucleotide sequence of the lyase glyssX609 is as shown in SEQ ID NO.1. The lyase lyssX609 provided by the invention has the advantages of strong stability and high lysing efficiency, and has a lysing effect on a plurality of Gram-positive bacteria.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a lysase plyssX609 for lysing Gram-positive bacteria and its application. Background technique [0002] Streptococcus is an important zoonotic pathogen, generally hemolytic facultative anaerobic Gram-positive bacteria with biofilm. Clinically, it has strong pathogenicity, mainly including Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae and so on. The disease caused by streptococcal infection is a serious threat to the animal husbandry production industry. Suis streptococcal disease is a zoonotic disease caused by Streptococcus suis pathogen, which is a legal second-class infectious disease in my country. It mainly causes meningitis, arthritis and endocarditis in piglets. According to the different capsular serotypes, it is currently divided into 29 kinds of streptococci. Streptococcus suis type 2 has a strong pathogenicity, and types 1, 2, 7 and 9 a...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K38/51A61P31/04C12R1/19
CPCC12N9/88C12N15/70A61P31/04A61K38/00
Inventor 钱平李祥敏王爽李鑫鑫
Owner 武汉道前生物科技有限公司
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