Phage lytic enzymes as well as gene, gene recombinant expression vector and application thereof

A technology of bacteriophage lyase and expression vector, applied in the field of bacteriophage lyase and its gene, gene recombinant expression vector and application field, can solve the problem of lack of effective prevention and treatment of drug-resistant bacterial infection, etc., and achieve good lysing bacterial activity Effect

Active Publication Date: 2020-12-29
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of the above deficiencies in the prior art, the purpose of the present invention is to provide a phage lyase and its gene, gene recombinant ex

Method used

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  • Phage lytic enzymes as well as gene, gene recombinant expression vector and application thereof
  • Phage lytic enzymes as well as gene, gene recombinant expression vector and application thereof
  • Phage lytic enzymes as well as gene, gene recombinant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning of Phage Lyase P9ly Gene

[0037] 1. Amplification of the lyase gene (using the lytic phage PDS9 genomic DNA corresponding to Shigella BDS9 as a template).

[0038](1) The primer sequences used for the amplification of phage PDS9 lyase gene P9ly include SEQ ID NO: 3 and SEQ ID NO: 4, wherein, SEQ ID NO: 3 is a forward primer: 5'-CATG CCATGG CAATGGATATTTTTTGATATGTTACG-3'; SEQ ID NO: 4 is reverse primer: 5'-CG GAATTC TCTATAAGCAGCCCATGTGC-3'. Wherein, the underlined part of the forward primer and the reverse primer represent NcoI and EcoRI restriction sites, respectively.

[0039] (2) The amplification reaction system is as follows:

[0040] Table 1 Components of PCR amplification reaction system

[0041]

[0042]

[0043] (3) The amplification conditions are as follows:

[0044] Mix the reaction system evenly, pre-denature at 94°C for 10min, then denature at 94°C for 45s, anneal at 58°C for 45s, extend at 72°C for 90s, and after 30 cycles, extend at 7...

Embodiment 2

[0051] Construction of recombinant expression vector pET28a-P9ly

[0052] In order to connect the target gene fragment to the expression vector pET28a, it is necessary to make the target fragment have a fragment with cohesive ends, that is, a restriction site.

[0053] 1. Preparation of linear vector pET28a with cohesive ends

[0054] In order to connect the target gene fragment to the expression vector pET28a, it is necessary to make the target fragment have a fragment with cohesive ends, that is, a restriction site. Similarly, in order to allow the target fragment to be inserted into the vector, it is also necessary to make the vector have cohesive ends and make their restriction sites the same.

[0055] (1) Plasmid extraction: Use the plasmid extraction kit (Bitec), the operation steps are as follows:

[0056] ① Strain activation: Dip the sterile inoculation loop into the strain preservation solution frozen at -80°C, and inoculate it on the Kan + LB plate, cultivate over...

Embodiment 3

[0074] Induced Expression and Enzyme Activity Determination of Lyase P9ly Recombinant Protein in Escherichia coli BL21(DE3)

[0075] The constructed recombinant expression vector pET28a-P9ly was transformed into Escherichia coli BL21(DE3), the strain containing the recombinant plasmid was cultured overnight, and the bacterial solution was inoculated into Kan + (final concentration 50 μg / mL) of LB liquid medium, 37 ° C shaker culture to its OD value of 0.6-0.8; take out 4 mL of bacterial liquid as a control experiment; add lactose (final concentration of 1 mg / mL) to the remaining bacterial liquid ), placed at 37° C., 150 rpm shaker for induction culture for 10 hours, and sampled 5 mL.

[0076] 1. Lyase P9ly protein SDS-PAGE detection

[0077] Centrifuge the 5mL bacterial liquid taken out at 9000rpm for 10min, discard the supernatant, add a final concentration of 30mM imidazole solution to suspend the bacterial cells, ultrasonically disrupt the bacterial cells (power 36%, beat ...

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Abstract

The invention discloses phage lytic enzymes as well as a gene recombinant expression vector and application thereof. The phage lytic enzyme expression gene is a nucleotide sequence shown as SEQ ID NO:1, and the nucleotide sequence can express phage lytic enzymes in a host cell by constructing a recombinant vector. The phage lytic enzymes obtained through purification have relatively high in-vitroand in-vivo bactericidal activity, acts quickly, is safe and harmless to organisms, can well treat multi-drug-resistance shigella infection, and also has a certain inhibition effect on staphylococcusaureus, vibrio parahaemolyticus and other pathogenic bacteria. The phage lytic enzymes have good lytic thallus activity within the range of 28-42 DEG C, and due to the characteristics, the phage lytic enzymes have a wide prospect in the aspect of preparing drug-resistant infectious disease medicaments and can be used as a substitute or supplement of conventional antibiotics.

Description

technical field [0001] The invention relates to the technical field of phage application, in particular to a phage lyase and its gene, gene recombination expression vector and application. Background technique [0002] Since the introduction of antibiotics in clinical practice in the 1940s, they have played a huge role in the treatment of bacterial infections in humans and animals. However, with the overuse of antibiotics, leading to the rapid emergence and spread of uncontrolled resistance determinants in almost all bacterial pathogens, many current bacterial infections have become highly refractory to the vast majority of traditional antibiotics. [0003] Bacterial drug resistance refers to the insensitivity of bacteria to various antibacterial drugs, that is, the resistance to foreign antibacterial drugs, making their curative effect worse or even lose their effect. Bacterial drug resistance is mainly divided into two types: one is genetically mediated, called intrinsic ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21A61K38/51A61P31/04C12R1/19
CPCC12N9/88C12N15/70A61P31/04A61K38/00Y02A50/30
Inventor 王峰肖瑶林连兵邓征宇邓先余张棋麟
Owner KUNMING UNIV OF SCI & TECH
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