A light-responsive engineered bacteria intestinal targeting optogenetic carrier system and its construction method and application

Abstract

The invention relates to a light-responsive engineering bacteria gut-targeted optogenetic carrier system. The system is a blue-light-responsive engineering bacteria BL21@pH-sensitive microsphere-carrying rare earth nanomaterial@acid-alkaline-enzyme-stabilized two-component microspheres The two-component microspheres are engineering bacteria BL21@alginate microspheres loaded with rare earth nanomaterials@acid-alkaline-enzyme stable microspheres uniformly coated with chitosan. The light-responsive engineering bacteria intestinal optogenetic carrier system of the present invention is a blue light-responsive engineering bacteria BL21@pH-sensitive microsphere-carrying rare earth nanomaterial@acid-base-enzyme-stabilized two-component microspheres, with uniform morphology and good dispersibility , which can target inflammatory bowel disease lesions and simultaneously release blue-light-responsive engineered bacteria BL21 and rare earth nanomaterials@acid-alkaline-enzyme-stabilized microspheres, using "optogenetic technology" to achieve precise and effective colonization of exogenous engineered bacteria, further To achieve the relief and treatment of inflammatory bowel disease.

Description

technical field

[0001] The invention belongs to the fields of biotechnology and medical technology, in particular to a light-responsive engineered bacteria gut-targeting optogenetic carrier system, construction method and application. Background technique

[0002] Clinical applications show that recombinant engineered bacteria transplantation plays an indispensable role in the prevention, mitigation and treatment of Clostridium difficile infection, inflammatory bowel disease, diabetes, cancer, liver cirrhosis and related encephalopathy. It has been reported that 90% of clinical symptoms of Clostridium difficile infection can be alleviated by colonization with recombinant engineered bacteria; in animal models of Parkinson's disease, recombinant engineered bacteria transplantation also plays an important role in regulating motor deficits and neuroinflammation. In addition, compared with oral or injectable drugs, bacteria, as endogenous "drug factories" in the gut, are more sui...

Examples

preparation example Construction

[0052] The preparation method of the above-mentioned carrier system, the concrete method is as follows:

[0053] (1) Preparation of rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres: Weigh rare earth nanomaterials (such as blue upconversion rods, which can be referred to as upconversion rods) and dissolve them in the prepolymer solution. The ratio g:mL is 0.1-0.5:1-5, and the composition ratio of the prepolymer solution is: polyethylene glycol diacrylate-200: polyethylene glycol-700 (wherein, 200 and 700 are the molecular weights of these two substances ): the volume ratio of 2-hydroxy-2-methyl-1-phenyl-1-acetone is 5:4-3:1-2, when the prepolymer solution is prepared, the polyethylene glycol diacrylate- 200: polyethylene glycol-700: 2-hydroxy-2-methyl-1-phenyl-1-acetone mixed in proportion; ultrasonically dispersed, stored in the dark, as the internal solution; dimethyl silicone oil as the external solution. Then prepare rare earth nanomaterials@acid-alkali-...

Embodiment 1

[0067] Preparation of rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres: Weigh 0.4g of rare earth nanomaterial rods and dissolve them in 2mL of prepolymerization solution. The composition ratio of the prepolymerization solution is PEGDA-200:PEG-700:HMPP=5:3 :2. Ultrasonic dispersion, protected from light, as an internal solution. Dimethicone was used as the external liquid. Relying on the microfluidic platform built by the Nanotechnology Laboratory, School of Life Sciences, Tianjin University, rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres with uniform particle size were prepared.

Embodiment 2

[0069] Construction of blue light-responsive E. coli BL21 engineering strain: PCR technology was used to amplify the pDawn-Ag43 fragment from the template (Addgene#107742). The vector pUC19 was linearized by PCR technology, and the original promoter region was avoided. Using homologous recombination, the pDawn-Ag43 fragment was ligated with the pUC19 vector, and the constructed plasmid pUC19-pDawn-Ag43 was first transformed into a DH5a competent strain and sequenced to obtain a recombinant-positive plasmid. On this basis, the E. coli BL21 expression plasmid pUC19-pDawn-Ag43-TGF-β1 containing transforming growth factor (TGF-β1) immune factor was constructed.

technical field

[0001] The invention belongs to the fields of biotechnology and medical technology, in particular to a light-responsive engineered bacteria gut-targeting optogenetic carrier system, construction method and application. Background technique

[0002] Clinical applications show that recombinant engineered bacteria transplantation plays an indispensable role in the prevention, mitigation and treatment of Clostridium difficile infection, inflammatory bowel disease, diabetes, cancer, liver cirrhosis and related encephalopathy. It has been reported that 90% of clinical symptoms of Clostridium difficile infection can be alleviated by colonization with recombinant engineered bacteria; in animal models of Parkinson's disease, recombinant engineered bacteria transplantation also plays an important role in regulating motor deficits and neuroinflammation. In addition, compared with oral or injectable drugs, bacteria, as endogenous "drug factories" in the gut, are more sui...

Examples

preparation example Construction

[0052] The preparation method of the above-mentioned carrier system, the concrete method is as follows:

[0053] (1) Preparation of rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres: Weigh rare earth nanomaterials (such as blue upconversion rods, which can be referred to as upconversion rods) and dissolve them in the prepolymer solution. The ratio g:mL is 0.1-0.5:1-5, and the composition ratio of the prepolymer solution is: polyethylene glycol diacrylate-200: polyethylene glycol-700 (wherein, 200 and 700 are the molecular weights of these two substances ): the volume ratio of 2-hydroxy-2-methyl-1-phenyl-1-acetone is 5:4-3:1-2, when the prepolymer solution is prepared, the polyethylene glycol diacrylate- 200: polyethylene glycol-700: 2-hydroxy-2-methyl-1-phenyl-1-acetone mixed in proportion; ultrasonically dispersed, stored in the dark, as the internal solution; dimethyl silicone oil as the external solution. Then prepare rare earth nanomaterials@acid-alkali-...

Embodiment 1

[0067] Preparation of rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres: Weigh 0.4g of rare earth nanomaterial rods and dissolve them in 2mL of prepolymerization solution. The composition ratio of the prepolymerization solution is PEGDA-200:PEG-700:HMPP=5:3 :2. Ultrasonic dispersion, protected from light, as an internal solution. Dimethicone was used as the external liquid. Relying on the microfluidic platform built by the Nanotechnology Laboratory, School of Life Sciences, Tianjin University, rare earth nanomaterials@acid-alkali-enzyme stabilized microspheres with uniform particle size were prepared.

Embodiment 2

[0069] Construction of blue light-responsive E. coli BL21 engineering strain: PCR technology was used to amplify the pDawn-Ag43 fragment from the template (Addgene#107742). The vector pUC19 was linearized by PCR technology, and the original promoter region was avoided. Using homologous recombination, the pDawn-Ag43 fragment was ligated with the pUC19 vector, and the constructed plasmid pUC19-pDawn-Ag43 was first transformed into a DH5a competent strain and sequenced to obtain a recombinant-positive plasmid. On this basis, the E. coli BL21 expression plasmid pUC19-pDawn-Ag43-TGF-β1 containing transforming growth factor (TGF-β1) immune factor was constructed.

Images