Bacteriophage lytic enzymes as alternative antimicrobials

一种噬菌体裂解酶、抗微生物的技术，应用在作为可替代的抗微生物剂的噬菌体裂解酶领域

Inactive Publication Date: 2013-12-11
US SEC AGRI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Without the traditional antibiotics used to prevent necrotizing enteritis and other diseases caused by Clostridium perfringens, these diseases could become a huge problem for the poultry industry

Method used

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  • Bacteriophage lytic enzymes as alternative antimicrobials
  • Bacteriophage lytic enzymes as alternative antimicrobials
  • Bacteriophage lytic enzymes as alternative antimicrobials

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0147] The following example shows phage lytic isolation of Clostridium perfringens.

[0148] Bacterial hosts, phage isolation and propagation

[0149] Clostridium perfringens isolates 39 and 26 were used as strains obtained from 0.45 μm filtered offal wash (O) or chicken manure (F) at a chicken processing facility in Athens, GA. Host for phage proliferation. Host strains were characterized by standard methods and 16S sequence analysis (Collins et al., 1994; Wise and Siragusa, 2005). Initial screens for phage lysis of Clostridium perfringens were performed on filtered samples obtained from poultry (gut material), soil, sewage and poultry processing effluent water. Bacterial viruses capable of lysing strains of Clostridium perfringens were identified by sampling and titration of isolated phage-susceptible strains. Clostridium perfringens was propagated by standard procedures (Ackermann and Nguyen, 1983) using anaerobic chamber-grown C. perfringens isolates 39 and 26 ho...

Embodiment 2

[0152] The following examples illustrate molecular cloning, sequencing, annotation and phylogenetic analysis of genomic DNA.

[0153] Following purification of phage genomic DNA, nucleic acids were read spectrophotometrically and restriction enzyme digested at 260 / 280 nm, followed by agarose gel electrophoresis to assess purity (Sambrook et al., supra). Full-length genome sequencing of the phage genome was performed by MWG Biotech, Inc High Point, NC USA. Briefly, phage genomic DNA was sheared, blunt-ended and dephosphorylated using a sprayer (Sambrook et al., 1989). After transformation, blunt-end ligate DNA fragments of the desired size (1 to 4 kb) into pSmart (Lucigen TM ) for the proliferation of Escherichia coli. Clones were sequenced such that approximately 14-fold redundancy was obtained for the genome including primer walking to fill gaps (Fouts et al., 2006). restriction enzyme Hin dIII, Eco RI, Eco RV, Alu I and cla I perform molecular cloning to cut the...

Embodiment 3

[0156] The following example shows virion purification, two-dimensional electrophoresis and proteomic analysis of isolated CP39O (PTA-11081) and CP26F (PTA-11080) phage.

[0157] Purified virion sample preparation for two-dimensional (2D) gel electrophoresis. Phage protein purification was accomplished by adding four volumes of cold (-20°C) acetone to pellet the centrifuged phage for at least one hour. Centrifuge the soluble protein fraction in acetone at 16,000 x g for 10 min at 4 °C. The pellet was washed three times in cold acetone / water (4:1 ), then centrifuged, and dried under vacuum (Champion et al., 2001; Lee and Lee, 2003). To ensure reproducible gel electrophoresis, gel electrophoresis was performed at 37°C according to the manufacturer's instructions (New England BioLab TM ) The purified virion protein fraction was digested with N-glycosidase F (PNGase F) for 1 hr to cleave oligosaccharides from N-linked glycoproteins (Maley et al., 1989) prior to extraction with ...

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Abstract

The present invention relates to isolated Clostridium perfringens bacteriophage lytic enzymes from baccteriophages CP26F and CP39O, and uses in controlling Clostridium perfringens.

Description

technical field [0001] The present invention relates to be used for controlling Clostridium perfringens ( Clostridium perfringens ) bacterial phage lytic enzyme or functional fragment thereof and uses of said lytic enzyme, including but not limited to compositions and methods for treating diseases caused by Clostridium perfringens. Background technique [0002] Clostridium perfringens is a Gram-positive, rod-shaped, anaerobic, spore-forming bacterium of the genus Clostridium. Clostridium perfringens is ubiquitous in nature and is commonly found as components of decaying plants, marine sediments, guts of humans and other vertebrates, insects and soils. Although ubiquitous and often benign, Clostridium perfringens is also the cause of serious infections in many animals and humans. Indeed, Clostridium perfringens is known to be the cause of food poisoning, gas gangrene (clostridial myonecrosis), necrotizing enteritis, and non-food borne gastrointestinal infections. Clostridi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/36C12N15/56A61K38/47A61P31/04A23K1/165C12R1/145
CPCA23K20/189A23K50/75A61P31/04C12N9/80C12N2795/10321C12N2795/10322C12N2795/10332C12Y305/01028
Inventor 布鲁斯·S·希尔格雷戈里·R·希拉古萨伊本·穆斯塔法·A·西蒙斯乔娜·K·加利什大卫·M·多诺万
Owner US SEC AGRI
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