Vibrio parahaemolyticus bacteriophage lyase after gene engineering transformation and preparation method and application of vibrio parahaemolyticus bacteriophage lyase

A technology of phage lyase and hemolytic vibrio, which is applied in the field of genetic engineering, can solve problems such as hindering the cleavage of phage lyase, and achieve the effect of improving stability

Active Publication Date: 2020-06-19
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Phage lyase mainly acts on the peptidoglycan on the bacterial cell wall, so it has a significant lytic effect on Gram-positive bacteria; but for Gram-negative bacteria, the lipopolysaccharide outer membrane of the bacterial cell wall hinders phage lyase play a lytic role

Method used

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  • Vibrio parahaemolyticus bacteriophage lyase after gene engineering transformation and preparation method and application of vibrio parahaemolyticus bacteriophage lyase
  • Vibrio parahaemolyticus bacteriophage lyase after gene engineering transformation and preparation method and application of vibrio parahaemolyticus bacteriophage lyase
  • Vibrio parahaemolyticus bacteriophage lyase after gene engineering transformation and preparation method and application of vibrio parahaemolyticus bacteriophage lyase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Example 1 Amplification of connecting peptide 1 connecting PCNP-lys target fragment

[0071] Step 1, using the Vibrio phage lysing enzyme lys gene (the number in its NCBI protein database is ADX87518.1, and the nucleotide sequence is shown in SEQ ID NO: 20) as a template, use primer 4 and primer 5 to carry out the first PCR, to obtain the target fragment connected with a connecting peptide 1 and the second half of PCNP, and recover the PCR product as the template for the next amplification; then use primer 5 and primer 3 for the second PCR, and obtain a connecting peptide 1 and a complete PCNP target fragment (the purpose of this PCR is mainly to connect the first half of PCNP), and the PCR product is recovered as a template for the next amplification; then the third PCR is carried out with primer 2 and primer 5, and a complete connection is obtained. PCNP and two new target fragments of the connecting peptide 1 (the purpose of this PCR is to connect the connecting pept...

Embodiment 2

[0074] Example 2 Connecting peptide 2 to connect PCNP-lys target fragment amplification

[0075] Step 1, using the lys gene of Vibrio phage lyase (the number in its NCBI protein database is ADX87518.1, and the nucleotide sequence is shown in SEQ ID NO: 20) as a template, use primer 12 and primer 5 to carry out the first PCR, to obtain the target fragment connected with a connecting peptide 2 and the second half of PCNP, and recover the PCR product as a template for the next amplification; then use primer 5 and primer 11 for the second PCR, and obtain a connecting peptide 2 and a target fragment of a complete PCNP (the main purpose of this PCR is to connect the first half of PCNP) to recover the PCR product as a template for the next amplification; then use primer 10 and primer 5 to carry out the third PCR, and obtain a connection with complete PCNP and Two new target fragments of connecting peptide 1 (the purpose of this PCR is to connect connecting peptide 2 for the second ti...

Embodiment 3

[0078] Example 3 Amplification of the PCNP-lys target fragment connected without connecting peptide

[0079] Step 1, using the Vibrio phage lysing enzyme lys gene (the number in its NCBI protein database is ADX87518.1, and the nucleotide sequence is shown in SEQ ID NO: 20) as a template, use primer 8 and primer 5 to carry out the first PCR, to obtain the target fragment connected to the second half of PCNP, reclaim the PCR product as the template for next amplification; then use primer 5 and primer 7 to carry out the second PCR, and obtain the target fragment connected to a complete PCNP (this PCR Mainly connected to the first half of PCNP); the PCR product was recovered as the template for the next amplification.

[0080] Step 2, repeating the step of step 1 successively, obtained the target segment connecting two PCNPs (connecting a new PCNP target segment);

[0081] Step 3, finally use primer 6 and primer 5 to carry out the last PCR (the purpose is to introduce the restric...

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Abstract

The invention provides vibrio parahaemolyticus bacteriophage lyase PCNP-lys, and the amino acid sequence of the PCNP-lys is shown in SEQ ID NO:1. The invention further provides an antibacterial agentwhich comprises main active components of the vibrio parahaemolyticus bacteriophage lyase PCNP-lys, a carrier with a PCNP-lys expression element, an expression cassette with the PCNP-lys expression element or at least one of host cells with the PCNP-lys expression element. The invention further provides a preparation method of the vibrio parahaemolyticus bacteriophage lyase PCNP-lys. The vibrio parahaemolyticus bacteriophage lyase PCNP-lys provided by the invention is capable of splitting multiple pathogenic vibrios, and makes a basis for substitution therapies of drug-resistant vibrio parahemolyticus.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a genetically engineered vibrio parahaemolyticus phage lyase and its preparation method and application. Background technique [0002] Vibrio parahaemolyticus, one of the most common pathogenic species of the Vibrio genus, is a Gram-negative, halophilic spore-forming bacterium with a straight and single rigid curve, having a single polar flagella , can move when grown in liquid medium. This bacterium is a human pathogen that occurs naturally in the marine environment and is frequently isolated from a variety of seafood, including cod, sardines, crayfish, flounder, mackerel, clams, octopus, shrimp, crab, scallops, and oysters . [0003] According to the Food and Agriculture Organization, aquaculture is one of the fastest growing sectors of animal food production, supporting approximately 50% of global human and fish consumption. Consumption of raw or undercooked s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56A61K38/47A61P31/04A23K50/80A23K20/189
CPCA61K38/00A23K20/189A23K50/80A61P31/04C12N9/2402C12Y302/01
Inventor 田园马兴元
Owner EAST CHINA UNIV OF SCI & TECH
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