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Application of bacteriophage endolysins Lysep3 in preparation of broad-spectrum antibacterial drug

A technology of phage lysing enzymes and antibacterial drugs, which is applied in the fields of biomedicine and genetic engineering, can solve the problems of high production cost of natural lysing enzymes, unclear bactericidal effect and mechanism, and achieve important application value and broad market prospects

Active Publication Date: 2020-07-17
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
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Problems solved by technology

However, the lyases that have been isolated and identified are still not enough to meet the needs of animal husbandry production. At the same time, the production cost of natural lyases is relatively high, and the bactericidal effect and mechanism are not clear. Therefore, the production process of natural lyases is optimized and modified. Further research on its application field is also the difficulty and hope of the current lyase product development

Method used

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  • Application of bacteriophage endolysins Lysep3 in preparation of broad-spectrum antibacterial drug
  • Application of bacteriophage endolysins Lysep3 in preparation of broad-spectrum antibacterial drug
  • Application of bacteriophage endolysins Lysep3 in preparation of broad-spectrum antibacterial drug

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The acquisition of embodiment 1 lyase Lysep3 gene fragment

[0065] Optimization and design of lyase Lysep3 gene expression sequence:

[0066] The gene encoding the lyase Lysep3 was optimized according to the yeast codon preference (SEQ ID NO:2), and 8 CAT histidine tags and 2 TAA terminator sequences were added to the 3' end of the optimized gene sequence (SEQ ID NO:2). ID NO:3), and add XhoI restriction site and Kex2 restriction site at the 5' end of the optimized gene sequence by the method of specific primer PCR amplification, and add XbaI restriction site at the 3' end, that is Complete the construction of the gene expression cassette. The above sequence was completed by Shanghai Sangon Bioengineering Co., Ltd.

Embodiment 2

[0067] Example 2 Construction of Yeast Recombinant Expression Vector

[0068] 1. Amplify the lyase Lysep3 gene fragment obtained in Example 1 with specific primers containing the XhoI and XbaI restriction site sequences, and perform double digestion to recover the purified fragment. At the same time, the pPICZαA vector (purchased from Invitrogen) was digested with XhoI and XbaI, and the product was verified by Tricine-SDS-PAGE electrophoresis ( figure 1 ).

[0069] The double enzyme digestion system is as follows:

[0070]

[0071] After adding the sample to the above enzyme digestion system, place it in a PCR instrument at 37°C for 4 hours, and detect it by electrophoresis on a 2% agarose gel. Electrophoresis conditions: 120V, 30min. The digested products were recovered with a DNA product recovery kit. After the lyase Lysep3 gene and the pPICZαA vector were digested with XbaI and XhoI, the lyase Lysep3 gene was ligated with the linearized pPICZαA vector with T4 DNA liga...

Embodiment 3

[0088] Example 3 Construction of recombinant yeast strain containing lyase Lysep3 gene

[0089] 1. Linearization of recombinant vector pPICZαA-Lysep3

[0090] Use PmeI to digest the constitutive recombinant expression vector pPICZαA-Lysep3. The enzyme digestion system and reaction conditions are as follows:

[0091]

[0092] After adding the sample to the above enzyme digestion system, place it in a PCR instrument at 37°C for 4 hours, and detect it by electrophoresis on a 2% agarose gel. Electrophoresis conditions: 120V, 30min. Electrophoresis results showed that the pPICZαA-Lysep3 recombinant vector was completely linearized.

[0093] 2. Preparation of competent Pichia pastoris X-33

[0094] 1) Pick a single colony of X-33 on the YPD plate, inoculate it into 10 mL of YPD liquid medium, and cultivate overnight at 29°C and 250 rpm;

[0095] 2) Take Pichia pastoris X-33 overnight culture solution and inoculate it into 100mL YPD liquid medium with 1% inoculum amount, cultiv...

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Abstract

The invention provides an application of bacteriophage endolysins Lysep3 in preparation of a broad-spectrum antibacterial drug. The invention finds for the first time that the Lysep3 has a better inhibitory effect on gram-positive bacteria, and has an obvious inhibitory effect on a plurality of bacteria under the combined action of an outer membrane penetrant, and has lower hemolytic activity on mouse red blood cells and cytotoxicity to murine derived macrophages; and in addition, the natural bacteriophage endolysins Lysep3 is used as a template, a specific expression vector is constructed byoptimizing an endolysins Lysep3 gene sequence, so that the expression of the bacteriophage endolysins Lysep3 in an eukaryotic expression system (pichia pastoris) is realized, a protein purification system is established and perfected, the large-scale production of the bacteriophage endolysins Lysep3 can be realized, and the bacteriophage endolysins Lysep3 is used in the fields of antibacterial drugs and feed additives, and has important application value and broad market prospects.

Description

technical field [0001] The invention relates to the fields of biomedicine and genetic engineering, in particular to the application of phage lyase Lysep3 in the preparation of broad-spectrum antibacterial drugs. Background technique [0002] China is a big country in the production and consumption of livestock and poultry products, and it is also a big country in feed production and antibiotic use in the world. In 2010, the consumption accounted for about 23% of the global consumption, and it may increase to 30% by 2030 (Van Boeckel et al., 2015 ). The use of antibiotics in animal husbandry has played an important role in promoting the development of the aquaculture industry, but the use of a large number of antibiotics has also led to the proliferation of drug resistance of pathogenic bacteria in the animal husbandry industry. Studies have shown that, especially in underdeveloped countries and regions such as Asia, one-third of commonly used drugs are ineffective against c...

Claims

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Application Information

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IPC IPC(8): A61K38/47A61P31/04C12N9/24C12N15/81C12N1/19C12R1/84
CPCA61K38/47A61P31/04C12N9/2402C12Y302/01C12N15/815Y02A50/30
Inventor 王建华闫伟滕达毛若雨杨娜王秀敏郝娅马炫炫
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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