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Clostridium perfringensphage lyase and application thereof

A technology of Clostridium perfringens and phage lyase, which is applied in the field of bioengineering and can solve the problems affecting the clinical treatment effect of antibiotics, the decomposition of carbohydrates in vascular endothelial cells, and tissue edema.

Active Publication Date: 2021-05-28
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the widespread use of antibiotics, the resistance of Clostridium perfringens to antibiotics is increasing, and the phenomenon of multi-drug resistance is obvious, which seriously affects the clinical effect of antibiotics.
Poultry is the main host of Clostridium perfringens. It is often contaminated by Clostridium perfringens during slaughtering and processing. However, eating food contaminated by Clostridium perfringens can cause food poisoning, which can damage cell membranes and blood vessels. Endothelial cells decompose sugars, leading to cell necrosis, tissue edema, inflation and other lesions, and even death in severe cases

Method used

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  • Clostridium perfringensphage lyase and application thereof
  • Clostridium perfringensphage lyase and application thereof
  • Clostridium perfringensphage lyase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Phage gene extraction

[0025] Filter the supernatant of the phage vB_CpeS_JS01 lysate with a 0.22 μm filter membrane, add RNaseA and DNase I to the phage suspension at a final concentration of 1 μg / ml, incubate at 37°C for 30 minutes; add 9.3g PEG 8000, 5.8g NaCl, shake well until dissolved , ice bath for 1h; centrifuge at 10000×g min at 4°C -1 , 30min, remove the supernatant; add 5ml SM solution, fully wash the tube wall and precipitate, and react at room temperature for 30min; add an equal volume of chloroform to extract PEG and cell fragments in the phage suspension, shake for 30s; 4℃, 3000×g min -1 Centrifuge for 15 min, recover the hydrophilic phase containing phage particles, and obtain purified phage.

[0026] The prepared phage suspension was used to extract the phage genome with the lambda phage genome rapid extraction kit method, specifically according to the kit instructions.

Embodiment 2

[0028] Cloning of Lyase Cps-Z1 and Construction of Expression Vector

[0029] a. Design a pair of specific primers according to the coding sequence of the lyase nucleotide sequence (Seq ID NO.1):

[0030] CP-ZF:5’-ATT GGA TCC ATG CTG CTG ATT AAC TGC-3’

[0031] CP-ZR:5'-CTT AAG CTT TAA TTC AAC ACC TTT ATG TTT-3';

[0032] Use the phage genome as a template to amplify the full-length sequence of the Cps-Z1 gene with the above-mentioned specific primers, electrophoresis on 1% agarose, and identify the size of the amplified fragment;

[0033] b. Purify and recover the amplified target fragment by gel cutting, and connect the fragment with the pMD18-T vector overnight at 16°C, transform Escherichia coli DH5α competent cells the next day, and spread it on a plate containing ampicillin (100 μg / ml) , and the transformed positive clone was sequenced and identified, named as the lyase Cps-Z1 gene, and its nucleotide sequence was Seq ID NO.1;

[0034] c. Double digest the fragment wi...

Embodiment 3

[0038] Induced Expression and Purification of Lyase Cps-Z1 Protein

[0039] Inoculate the recombinant strain DE3 (pET-Cps-Z1) into LB medium containing kanapenicillin (100 μg / mL) and culture overnight at 37°C with shaking; the next day, transfer to 100mL LB medium at a ratio of 1:100 , shake culture at 37°C until the OD600 value is about 0.5, add IPTG to a final concentration of 1 mmol / L, and induce for 6 hours at 28°C. Bacteria were collected, cells were disrupted by ultrasonication, centrifuged at 10,000 rpm / min at 4°C for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane, and the protein expression in the lysed supernatant was analyzed by SDS-PAGE. The filtered lysed supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the instructions of the kit. The expression product Cps-Z1 was obtained, and the purified lyase Cps-Z1 product was detoxified (≤0....

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Abstract

The invention provides clostridium perfringensphage lyase and an application thereof serving as an antibacterial substance in prevention of poultry necrotic enteritis and food pollution control, and belongs to the field of bioengineering. The amino acid sequence of the clostridium perfringensphage lyase disclosed by the invention is SeqIDNO.2. The enzyme preparation capable of efficiently killing the clostridium perfringens is developed by utilizing a bioengineering technology, the preparation can be independently used or used in a compounded manner, the clostridium perfringens can be specifically inactivated, and a safe enzyme preparation source without toxic and side effects is provided for preventing and treating poultry necrotic enteritis and controlling clostridium perfringens pollution in food at present.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Clostridium perfringens phage lyase and its application. Background technique [0002] Clostridium perfringens (Cp) is an important zoonotic pathogen, which can not only cause necrotic enteritis (NE) in poultry, causing huge economic losses to the aquaculture industry, but also cause food poisoning and harm humans. Health poses a huge threat. With the widespread use of antibiotics, the resistance of Clostridium perfringens to antibiotics is increasing, and the phenomenon of multi-drug resistance is obvious, which seriously affects the clinical efficacy of antibiotics. Poultry is the main host of Clostridium perfringens. It is often contaminated by Clostridium perfringens during slaughtering and processing. However, eating food contaminated by Clostridium perfringens can cause food poisoning, which can damage cell membranes and blood vessels. Endothelial cel...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21A61K38/51A61P1/00A61P29/00C12R1/19
CPCC12N9/88C12N15/70A61K38/51A61P1/00A61P29/00Y02A50/30
Inventor 张辉包红朵周艳王冉庞茂达
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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