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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 LysYH6" in 2015, and Yang Hongjiang et al. from Tianjin University of Science and Technology applied for an invention patent "Pseudomonas and its application" (publication number CN107058265A), a bacteriophage lytic enzyme and bactericidal application (publication number CN105543256A) both adopt prokaryotic expression system, and its product separation and purification process is relatively complicated and not easy to scale up

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
Comparison scheme
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Embodiment 1

[0032] The purpose of this example 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 close to the Pseudomonas aeruginosa phage related to the present invention, and design a pair of primers according to the primer design principle, respectively LY23-F and LY23-R, the sequences of LY23-F and LY23-R are respectively shown in SEQ ID NO.3 and SEQ ID NO.4 in Table 1. Send the designed specific primers to Gene Synthesis Company for synthesis to obtain LY23-F and LY23-R respectively, and verify and confirm the obtained LY23-F and LY23-R by gene sequencing; amplify from Pseudomonas aeruginosa phage Cultivate, and use genome extraction kit (Qiagen company) to extract phage genome DNA, obtain DNA sample; Use high-fidelity polymerase Phusion (Thermo fisher company) to carry out polymerase chain reaction (PCR) am...

Embodiment 2

[0045] The purpose of this example 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. Digestion: The PCR recovery product in Example 1 and plasmids pSEP1, pSEP1alpha (ie vector pSEP1, pSEP1alpha) were double-digested with fast restriction endonucleases XhoI (Thermofisher Company) and EcoRI (Thermofisher Company) respectively. Take 3 EP tubes, numbered 1, 2, and 3. EP tubes numbered 1 and 2 were added to the components according to the reaction system in Table 5, and the plasmid DNA was the DNA of pSEP1 and pSEP1alpha (not transformed into LY23), Digested at 37°C for 10 minutes, and then terminated the reaction in a water bath at 80°C for 5 minutes to obtain the digested products of plasmid DNA (digested products of pSEP1 and pSEP1alpha). For PCR No. 3, add components according to the reaction system in Table 6. The fragment recovered by PCR was the purified target gene fr...

Embodiment 3

[0059] This embodiment provides a recombinant engineered bacterium comprising pSEP1-Ly23 and pSEP1alpha-Ly23.

[0060] A preparation process of a recombinant engineering bacterium comprising pSEP1-Ly23 and pSEP1alpha-Ly23 is as follows:

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

[0062] (1) Yeast cells were inoculated in YPD liquid medium and cultivated for 16-18 hours;

[0063] (2) collect the thalli, wash 3 times with ultrapure water;

[0064] (3) Finally, resuspend in a certain volume of PEG4000 (Chengdu Kelong Chemical Reagent Factory), so that the cell density is about 10 8 pc / mL, aliquoted into 250 μL / tube.

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

[0066] (1) Take two EP tubes, numbered 1 and 2, add 75 μL pSEP1-Ly23 carrier DNA and 250 μL β4 to the No. 1 EP tube, mix thoroughly, then add 40 μL 1M LiCl, and add 75 μl pSEP1alpha-Ly23 to the No. 2 EP tube Carrier DNA and 250 μL β4 were thoroughly mixed, and then ...

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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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