Beta-glucan gene vector, and preparation method and application thereof

Abstract

The invention discloses a beta-glucan gene vector, and a preparation method and application thereof. The preparation method comprises the following steps: (1) dissolving beta-glucan in a phosphate buffer solution, and heating at 130-145 DEG C for 15-60 minutes or dissolving in a dimethylsulfoxide or strongly alkaline solution to prepare a denatured single-chain polysaccharide solution; (2) bonding poly(dA)n with target nucleotide through a covalent bond, and dissolving in a buffer solution with the pH value of 7-9; and (3) adding the denatured single-chain polysaccharide solution in the step (1) into the poly(dA)n-target nucleotide / buffer solution, evenly mixing, and standing for 12-48 hours. The gene vector does not have cytotoxicity, has the advantages of favorable stability, higher transfection efficiency and higher immunocompetence, can avoid the inhibiting action of the serum due to the negative charge, can be widely used for CpGDNA transfection, can promote the release of inflammation regulatory factors, and can be used for immunotherapy.

Description

[0001] Technical field

[0002] The invention involves a β-glycosaccharide gene vector and its preparation methods and applications. It belongs to the field of high molecular physics and also belongs to the field of biomedical. Background technique

[0003] Gene therapy has become an effective treatment for many human diseases. Safe and effective gene vector is a key factor in the success of gene therapy.At present, most of the non -virus gene vectors, including cationic lipids, dendritic polymers, and nanoparticles have high cytotoxicity, which in turn limits the widespread application of these carriers.Only a few natural polymers, such as chitin and cellulose, are used for genetically transfection due to their good biocompatibility and low immunogenicity.However, the combination of chitin and cellulose and DNA is poor, resulting in lower transfusion efficiency.In order to solve these problems, it is necessary to chemically modify natural polymer to improve the efficiency of gen...

Examples

Embodiment 1

[0034] (1) Dissolve 0.1g of Lentinus edodes triple helical β-glucan (marked as t-LNT) in 1mL of dimethyl sulfoxide solvent to fully dissolve the denatured single-chain polysaccharide solution (marked as s-LNT).

[0035] (2) 5OD polydeoxyadenosine nucleotide (poly(dA) n ) through a covalent bond to 5OD target DNA (can be purchased from the company), and then dissolved in 0.01M, pH 7.4 phosphate buffer to prepare a DNA solution with a final concentration of 1 μg / μL.

[0036] (3) Add 4 μL of the poly(dA) obtained in (2) n - Add DNA / buffer solution to 194 μL of 0.01M, pH 7.4 phosphate buffer, then add 2 μL of the denatured single-chain polysaccharide solution obtained in (1) into the solution, mix well and let stand at 6°C for 48 hours. Complex gene vectors are available.

[0037] In this embodiment, the weight-average molecular weight of t-LNT is 4.8×10 4 (t-LNT1), 2.1×10 5 (t-LNT2), 5.7×10 5 (t-LNT3) and 7.5×10 5 (t-LNT4). Polydeoxyadenosine nucleotides are polydeoxynucle...

Embodiment 2

[0044] (1) The molecular weight of 0.01g is 2.1×10 5 The triple-helical β-glucan of shiitake mushrooms was dissolved in 1 mL of phosphate buffer (0.01M, pH 7.4) and fully dissolved, and then heated at 140°C for 30 minutes in a high-temperature and high-pressure reaction tube to prepare a denatured single-chain polysaccharide solution .

[0045] (2) 5OD polydeoxyadenosine nucleotide (poly(dA) n ) was bonded to 5OD target DNA (purchased from the company) through covalent bonds, and then dissolved in 0.01M, pH 7.4 phosphate buffer to prepare a DNA solution with a final concentration of 1 μg / μL. Among them, poly(dA) n Respectively poly(dA) 15 、poly(dA) 30 , and poly(dA) 50 ; The target DNA (DNA2) is a functional non-methylated phosphorothioate oligodeoxynucleotide (CpG DNA: 1826, 5'-TCCATGACGTTCCTGACGTT-3', 20 bases), used to investigate the success of DNA transfection and No; at the same time, the CpG DNA is transfected into the body to provide a scientific basis for gene i...

[0001] Technical field

[0002] The invention involves a β-glycosaccharide gene vector and its preparation methods and applications. It belongs to the field of high molecular physics and also belongs to the field of biomedical. Background technique

[0003] Gene therapy has become an effective treatment for many human diseases. Safe and effective gene vector is a key factor in the success of gene therapy.At present, most of the non -virus gene vectors, including cationic lipids, dendritic polymers, and nanoparticles have high cytotoxicity, which in turn limits the widespread application of these carriers.Only a few natural polymers, such as chitin and cellulose, are used for genetically transfection due to their good biocompatibility and low immunogenicity.However, the combination of chitin and cellulose and DNA is poor, resulting in lower transfusion efficiency.In order to solve these problems, it is necessary to chemically modify natural polymer to improve the efficiency of gen...

Examples

Embodiment 1

[0034] (1) Dissolve 0.1g of Lentinus edodes triple helical β-glucan (marked as t-LNT) in 1mL of dimethyl sulfoxide solvent to fully dissolve the denatured single-chain polysaccharide solution (marked as s-LNT).

[0035] (2) 5OD polydeoxyadenosine nucleotide (poly(dA) n ) through a covalent bond to 5OD target DNA (can be purchased from the company), and then dissolved in 0.01M, pH 7.4 phosphate buffer to prepare a DNA solution with a final concentration of 1 μg / μL.

[0036] (3) Add 4 μL of the poly(dA) obtained in (2) n - Add DNA / buffer solution to 194 μL of 0.01M, pH 7.4 phosphate buffer, then add 2 μL of the denatured single-chain polysaccharide solution obtained in (1) into the solution, mix well and let stand at 6°C for 48 hours. Complex gene vectors are available.

[0037] In this embodiment, the weight-average molecular weight of t-LNT is 4.8×10 4 (t-LNT1), 2.1×10 5 (t-LNT2), 5.7×10 5 (t-LNT3) and 7.5×10 5 (t-LNT4). Polydeoxyadenosine nucleotides are polydeoxynucle...

Embodiment 2

[0044] (1) The molecular weight of 0.01g is 2.1×10 5 The triple-helical β-glucan of shiitake mushrooms was dissolved in 1 mL of phosphate buffer (0.01M, pH 7.4) and fully dissolved, and then heated at 140°C for 30 minutes in a high-temperature and high-pressure reaction tube to prepare a denatured single-chain polysaccharide solution .

[0045] (2) 5OD polydeoxyadenosine nucleotide (poly(dA) n ) was bonded to 5OD target DNA (purchased from the company) through covalent bonds, and then dissolved in 0.01M, pH 7.4 phosphate buffer to prepare a DNA solution with a final concentration of 1 μg / μL. Among them, poly(dA) n Respectively poly(dA) 15 、poly(dA) 30 , and poly(dA) 50 ; The target DNA (DNA2) is a functional non-methylated phosphorothioate oligodeoxynucleotide (CpG DNA: 1826, 5'-TCCATGACGTTCCTGACGTT-3', 20 bases), used to investigate the success of DNA transfection and No; at the same time, the CpG DNA is transfected into the body to provide a scientific basis for gene i...

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