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Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture

A technology of fusion protein and spore wall, applied in the field of fusion protein, can solve the problems of affecting milk, not reducing the incidence of mastitis, pollution, etc.

Pending Publication Date: 2018-12-21
SPOGEN BIOTECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not only do they affect the collected milk (withdrawal for days, contamination with antibiotic residues, problems associated with yoghurt and cheese processing), antibiotics do not reduce the incidence of mastitis

Method used

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  • Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture
  • Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture
  • Fusion proteins, recombinant bacteria, and exosporium fragments for animal health and aquaculture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0850] Example 1. Use of various targeting sequences to express lipase on the surface of Bacillus thuringiensis

[0851] A wide variety of targeting sequences with high homology to amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1 ) can be used to display enzymes, proteins, and peptides on the surface of Bacillus cereus family members. Several targeting sequences were compared by making fusion proteins containing the targeting sequence linked to B. subtilis lipase. The fusion construct was synthesized using the native promoter of the targeting sequence, cloned into replicating plasmid pMK4, and introduced into Bacillus thuringiensis BT013A. Bacillus thuringiensis BT013A was deposited with the United States Department of Agriculture (USDA) Agricultural Research Service (ARS) on March 10, 2014 at 1815 North University Street, Peoria, Illinois 61604 USA, and assigned NRRL deposit number B-50924. Bacillus thuringiensis BT013A is also known as Bacillus thuringiensis 4Q7...

Embodiment 2

[0856] Example 2. Use of various exosporium sequences to express lipases on the surface of Bacillus thuringiensis and demonstrate localization of fusion proteins on the surface of the exosporium

[0857] A wide variety of exosporium proteins are available for displaying enzymes, proteins, and peptides on the surface of members of the Bacillus cereus family. Several different exosporium proteins were compared by preparing fusion proteins as described in Example 1 containing the exosporium protein linked to B. subtilis lipase. Fusion constructs were synthesized using the native promoter for exosporium proteins shown in Table 5 below, cloned into replicating plasmid pMK4, and introduced into B. thuringiensis BT013A. Spores displaying various exosporium protein-Bacillus subtilis 168 lipase fusions were plated on nutrient agar plates by growing transformed bacteria in brain heart infusion broth under selection pressure from 10 μg / ml chloramphenicol and incubated at 30°C for 3 days...

Embodiment 3

[0863] Example 3. Expression of endoglucanases on the surface of spores of members of the Bacillus cereus family using various targeting sequences

[0864] Cloning by homologous recombination PCR of the BclA promoter, start codon, and amino acids 20-35 of BclA (amino acids 20-35 of SEQ ID NO: 1 ) fused in-frame to the Bacillus subtilis 168 endoglucanase The resulting fragment was used to modify the pSUPER plasmid (pSUPER-BclA 20-35-Endo) (as described in Example 8 below). A PCR fragment was generated containing the BclA promoter (SEQ ID NO: 149) fused in frame to the Bacillus subtilis 168 endoglucanase, the start codon, and amino acids 20-35 of BclA. These PCR fragments were digested with XhoI and ligated into the SalI site of pSUPER plasmid to generate plasmid pSUPER-BclA 20-35-endoglucanase. This plasmid was then subjected to inverse PCR to amplify the entire plasmid backbone, but not the sequence corresponding to amino acids 20-35 of BclA. This inverse PCR product was com...

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Abstract

Fusion proteins, recombinant Bacillus cereus family members that express fusion proteins, and exosporium fragments derived from spores of the recombinant Bacillus cereus family members are provided. Compositions comprising the spores or exosporium fragments are also provided. Methods involving the use of spores of recombinant Bacillus cereus family members and exosporium fragments derived from spores of a recombinant Bacillus cereus family member in the fields of animal health and aquaculture are provided. In particular, methods are for provided for using such spores or exosporium fragments for protecting an animal or an aquatic organism from a pathogen. Methods are also provided for using exosporium fragments for producing an immunogenic response in an aquatic animal. Products for use inprotecting animals from pathogens are also provided, including adhesive patches, wound dressings, insert trays for livestock footbaths, hoof bandages, feed, feed additives, and insect foggers.

Description

technical field [0001] This application claims the benefit of US Provisional Application Serial No. 62 / 309,259, filed March 16, 2016, the entire contents of which are incorporated herein by reference. technical field [0002] The present invention relates generally to fusion proteins, recombinant Bacillus cereus family members expressing such fusion proteins, and exosporium fragments derived from spores of recombinant Bacillus cereus family members. The present invention also relates to compositions comprising spores of recombinant Bacillus cereus family members or exosporium fragments derived from spores of recombinant Bacillus cereus family members. Also provided are uses of spores of recombinant Bacillus cereus family members and exosporium fragments derived from spores of recombinant Bacillus cereus family members in the fields of animal health and aquaculture. In particular, the invention relates to methods of using spores or exosporium fragments to protect animals o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P1/04C12N11/16C07K14/32A61K39/00A61K39/02C12N15/74A01N63/50
CPCA61K39/07A61K39/385C07K14/32C12N15/75A61K2039/523A61K2039/552A61K2039/6068C07K2319/00C12R2001/085C07K14/325A61K39/0208A01N63/50A61K39/00A01N63/22A01N63/23A23K10/18A61F13/00063A61F2013/51047A61L2/18C02F3/348C12N1/20C12N3/00C12P21/02
Inventor B·M·汤普森A·西格尔
Owner SPOGEN BIOTECH INC
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