Pseudomonas aeruginosa bacteriophage lysin, coding gene thereof, recombinant expression vector as well as preparation method and application thereof

A technology of Pseudomonas aeruginosa and phage lysing enzymes, applied in the field of bioengineering, can solve the problems of not easy to scale up, complicated separation and purification process, complicated and time-consuming separation and purification process, and achieve low cost, strong cleavage activity, separation and purification simple effect

Inactive Publication Date: 2018-06-01
SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are few reports on the recombinant expression of Pseudomonas aeruginosa phage lyase, and the prokaryotic expression system is also used. The separation and purification process of the product is complicated and time-consuming, and the scale-up and production of pilot scale are difficult.
Yang Mei et al. from Jilin Agricultural University published the article "Expression and Activity of Pseudomonas aeruginosa Phage LysY

Method used

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  • Pseudomonas aeruginosa bacteriophage lysin, coding gene thereof, recombinant expression vector as well as preparation method and application thereof
  • Pseudomonas aeruginosa bacteriophage lysin, coding gene thereof, recombinant expression vector as well as preparation method and application thereof
  • Pseudomonas aeruginosa bacteriophage lysin, coding gene thereof, recombinant expression vector as well as preparation method and application thereof

Examples

Experimental program
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Example Embodiment

[0031] Example 1

[0032] The purpose of this embodiment is to provide a nucleotide sequence and amino acid sequence of a Pseudomonas aeruginosa phage lyase.

[0033] According to the homology and conservation of the species, select the DNA sequence of the Pseudomonas aeruginosa phage closely related to the Pseudomonas aeruginosa phage of the present invention, and design a pair of primers according to the primer design principles, respectively LY23-F And LY23-R, the sequences of LY23-F and LY23-R are shown in SEQ ID NO. 3 and SEQ ID NO. 4 in Table 1, respectively. The designed specific primers are sent to the gene synthesis company for synthesis, and LY23-F and LY23-R are obtained respectively, and the obtained LY23-F and LY23-R are confirmed by gene sequencing; amplified from Pseudomonas aeruginosa phage Culture, and use the genome extraction kit (Qiagen) to extract the phage genomic DNA to obtain a DNA sample; use the high-fidelity polymerase Phusion (Thermo fisher) for polymer...

Example Embodiment

[0044] Example 2

[0045] The purpose of this embodiment is to provide a recombinant expression vector of LY23.

[0046] The preparation process of a recombinant expression vector of LY23 is as follows:

[0047] 1. Enzyme digestion: The PCR product recovered in Example 1 was digested with plasmids pSEP1, pSEP1alpha (ie, vectors pSEP1, pSEP1alpha) with fast restriction enzymes XhoI (Thermo fisher company) and EcoRI (Thermofisher company) respectively. Take 3 EP tubes, numbered 1, 2 and 3. The EP tubes numbered 1 and 2 were added to each component according to the reaction system in Table 5. The plasmid DNA is the DNA of pSEP1 and pSEP1alpha (not transferred to LY23), Digestion at 37°C for 10 minutes, and stop the reaction in a water bath at 80°C for 5 minutes after the digestion is completed. The plasmid DNA digestion products (pSEP1 and pSEP1alpha digestion products) are obtained. The PCR number 3 adds the components according to the reaction system in Table 6. The PCR-recovered fr...

Example Embodiment

[0058] Example 3

[0059] This embodiment provides a recombinant engineered bacteria containing pSEP1-Ly23 and pSEP1alpha-Ly23.

[0060] The preparation process of a recombinant engineered bacteria containing pSEP1-Ly23 and pSEP1alpha-Ly23 is as follows:

[0061] 1. Preparation of yeast competent cells (β4):

[0062] (1) Inoculate yeast cells in YPD liquid medium and culture for 16-18 hours;

[0063] (2) Collect the bacteria and wash 3 times with ultrapure water;

[0064] (3) Finally, resuspend in a certain volume of PEG4000 (Chengdu Kelon Chemical Reagent Factory) to make the cell density about 10 8 Pieces / mL, divided into 250μL / tube.

[0065] 2. Transformation (introducing the recombinant expression vector into β4):

[0066] (1) Take 2 EP tubes, numbered 1 and 2. Add 75μL pSEP1-Ly23 vector DNA and 250μL β4 to EP tube 1 and mix well, then add 40μL 1M LiCl, and add 75μl pSEP1alpha-Ly23 to EP tube 2 Carrier DNA and 250μL β4 were mixed thoroughly and then 40μL 1M LiCl (Chengdu Kelong Chemic...

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Abstract

The invention belongs to the field of bioengineering technology, and discloses pseudomonas aeruginosa bacteriophage lysin, a coding gene thereof, a recombinant expression vector as well as a preparation method and an application thereof. An amino acid sequence of the pseudomonas aeruginosa bacteriophage lysin is shown as SEQ ID No.1, and a nucleotide sequence of the pseudomonas aeruginosa bacteriophage lysin is shown as SEQ ID No.2; the recombinant expression vector is constituted by recombining the nucleotide sequence of the pseudomonas aeruginosa bacteriophage lysin and a vector; and the recombinant expression vector is transformed into a host bacterium, so that a recombinant engineering bacterium is constituted. The pseudomonas aeruginosa bacteriophage lysin provided by the invention has the advantages that the pseudomonas aeruginosa bacteriophage lysin is relatively strong in lysis activity on pseudomonas aeruginosa in vitro. With the application of the preparation method of the pseudomonas aeruginosa bacteriophage lysin provided by the invention, the mass expression and preparation of the pseudomonas aeruginosa bacteriophage lysin can be achieved, and the preparation method issimple in separation and purification and is low in cost.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a pseudomonas aeruginosa phage lyase, its coding gene, a recombinant expression vector, its preparation method and application. Background technique [0002] The genus Pseudomonas is widely distributed in nature, and there are more than ten species that are pathogenic to humans and animals, among which Pseudomonas aeruginosa is the most representative. Pseudomonas aeruginosa belongs to Gram-negative bacteria and is a relatively common opportunistic pathogen. Many infections caused by Pseudomonas aeruginosa occur in hospitalized patients with compromised immunity or invasive procedures. It is one of the common pathogens of ICU infection and ventilator-associated pneumonia, and is the third most common pathogen causing nosocomial infections in patients. . In recent years, due to the overuse or abuse of antibiotics by humans, pathogenic bacteria have gradually de...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N15/81C12N1/21C12N1/19C12N15/11A61K38/51A61P31/04C12R1/19C12R1/84C12R1/865
CPCA61K38/00C12N9/88
Inventor 赵晨王辂李端华李进军葛燕
Owner SICHUAN INDAL INST OF ANTIBIOTICS CHINA NAT PHARMA GROUP CORP
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