CN103981201A - Beta-glucan gene vector, and preparation method and application thereof - Google Patents
Beta-glucan gene vector, and preparation method and application thereof Download PDFInfo
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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
Technical field
the present invention relates to a kind of beta-glucan genophore and its preparation method and application, it belongs to Polymer Physics field, also belongs to bio-medical field.
Background technology
Gene therapy has become effective therapeutic modality of mankind's numerous disease, and genophore is the key factor of gene therapy success or not safely and effectively.Most non-viral gene vectors of report, comprise that cationic-liposome, dendron shaped polymer and nanoparticle have higher cytotoxicity, and then have limited the widespread use of these carriers at present.Only has minority natural polymer, such as chitin and Mierocrystalline cellulose, because the advantages such as its good biocompatibility, reduced immunogenicity are used to gene transfection.But the binding ability of chitin and Mierocrystalline cellulose and DNA is poor, cause lower transfection efficiency.In order to address these problems, need carry out chemically modified to improve efficiency gene transfection to natural polymer.Beta-glucan has the natural polymer compared with high biological activity as another kind, especially, from the beta-glucan of yeast and fungi, can, by mouse and people's scavenger cell specific recognition, make beta-glucan have more advantage as novel medicine and genophore.
Summary of the invention
the shortcoming that the object of the invention is to overcome prior art, with not enough, provides a kind of beta-glucan genophore and its preparation method and application.
Object of the present invention is achieved through the following technical solutions:
A preparation method for beta-glucan genophore, comprises the steps:
(1) beta-glucan is dissolved in phosphate buffered saline buffer, at 130-145 ℃, heat 15-60 minute be dissolved in dimethyl sulfoxide (DMSO) or strong base solution in make the strand polysaccharide soln of sex change.
(2) will gather deoxyadenylic acid sequence (poly (dA)
n) by covalent linkage and target Nucleotide keyed jointing, be then dissolved in pH 7-9 damping fluid.
(3) the strand polysaccharide soln of the sex change (1) being obtained joins the poly (dA) that (2) obtain
nin-target Nucleotide/buffered soln, mix, standing 12-48 hour, obtains beta-glucan genophore.
Beta-glucan described in step (1) is preferably triple helical beta-glucan, and main chain is β-(1 → 3)-D-dextran, can contain the glucosyl residue of the different degrees of branching as side group; Described triple helical beta-glucan comprises curdlan, mushroom dextran, Sclerotium gum, Split-gill dextran, dictyophora fungus polysaccharide, mould granulose and xylan, and its molecular weight is preferably 5.0 * 10
4-4.5 * 10
5, different molecular weight triple helical beta-glucan can obtain by the method for ultrasonic (400-600W) degraded different time or the classification of acetone reprecipitation.
The concentration of the strong base solution described in step (1) is preferably > 0.1M.
Poly (dA) described in step (2)
nmiddle n preferably>=15.
Target Nucleotide described in step (2) comprises DNA and RNA, as CpG DNA, siRNA.
Poly (dA) used in step (2)
n, target nucleotide sequence Ke You company is synthetic provides, poly (dA)
ncomplete with target Nucleotide keyed jointing Ye You company, nucleic acid is dissolved in and in the damping fluid of pH7-9, is beneficial to that it is stable.
In step (3), the final concentration of the final concentration/nucleic acid of sex change strand polysaccharide preferably >=8.
In step (3), standing temperature is preferably 4-37 ℃.
A kind of beta-glucan genophore prepares by aforesaid method, and it can be used for reprinting of DNA and RNA etc., the especially reprinting of CpG DNA and siRNA.
Triple helical beta-glucan has antitumor, antiviral and anti-inflammatory biological immune activity preferably, and the mixture that itself and poly (dA) form has higher thermostability, resistance to hydrolysis and extremely low cytotoxicity.The present invention by the sex change of triple helical beta-glucan be after strand polysaccharide with poly (dA)
nthe beta-glucan genophore that-target Nucleotide forms is showed cell toxicity not, there is good stability, higher transfection efficiency, the electronegative property of while genophore, and can be widely used in the transfection of CpG DNA, and higher degree ground promotes the release of inflammation regulatory factor IL-12p40 and IL-6, for immunotherapy, further expand the functionalized application of triple helical beta-glucan.
Tool of the present invention has the following advantages and effect:
(1) triple helical beta-glucan wide material sources, output is abundant, with low cost, have no side effect, good biocompatibility.
(2) triple helical beta-glucan and poly (dA) have compared with strong interaction, have good stability and higher transfection efficiency.
(3) genophore that triple helical beta-glucan and poly (dA) build shows electronegativity, can avoid the restraining effect of serum.
(4) triple helical beta-glucan itself has immunocompetence, can, by mouse and people's scavenger cell specific recognition, for the transfection of CpG DNA, can be used for immunotherapy in vivo.
Accompanying drawing explanation
Fig. 1 is sex change triple helical beta-glucan and poly (dA)
nthe mixture genophore agarose gel electrophoresis figure that-DNA1 interacts and obtains; Wherein, in a, swimming lane 1,2,3,4 is respectively DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50, DNA1/s-LNT2, DNA1; In b, swimming lane 1,2,3,4,5 is respectively DNA1 – poly (dA)
50, DNA1 – poly (dA)
50/ s-LNT1, DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50/ s-LNT3, DNA1 – poly (dA)
50/ s-LNT4; In c, swimming lane 1,2,3,4,5,6 is respectively DNA1 – poly (dA)
15, DNA1 – poly (dA)
15/ s-LNT2, DNA1 – poly (dA)
30, DNA1 – poly (dA)
30/ s-LNT2, DNA1 – poly (dA)
50, DNA1 – poly (dA)
50/ s-LNT2; S-LNT1,2,3,4 represents to have the sex change mushroom triple helical beta-glucan sample of different molecular weight, and its molecular weight corresponds to respectively 4.8 * 10
4, 2.1 * 10
5, 5.7 * 10
5with 7.5 * 10
5.
Fig. 2 is that Laser Scanning Confocal Microscope qualitative analysis triple helical beta-glucan molecular weight is to beta-glucan/poly (dA)
50-DNA1 mixture genophore transfection efficiency affect result figure; Wherein, a-1, b-1, c-1, d-1 and e-1 are respectively DNA1 – poly (dA)
50, DNA1 – poly (dA)
50/ s-LNT1, DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50/ s-LNT3 and DNA1 – poly (dA)
50/ s-LNT4 cultivates altogether the burnt light field displaing micro picture of copolymerization after 48 hours in RAW264.7 cell; A-2, b-2, c-2, d-2 and e-2 are respectively DNA1 – poly (dA)
50, DNA1 – poly (dA)
50/ s-LNT1, DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50/ s-LNT3 and DNA1 – poly (dA)
50/ s-LNT4 cultivated altogether after 48 hours in RAW264.7 cell, the burnt picture of copolymerization under 405nm excitation (Hoechst 33258 dyeing for nucleus); A-3, b-3, c-3, d-3 and e-3 are respectively DNA1 – poly (dA)
50, DNA1 – poly (dA)
50/ s-LNT1, DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50/ s-LNT3 and DNA1 – poly (dA)
50/ s-LNT4 cultivated altogether after 48 hours in RAW264.7 cell, the burnt picture of copolymerization (gene transfection of cy3 mark is expressed) under 488nm excitation; A-4, b-4, c-4, d-4 and e-4 are respectively the burnt picture of copolymerization of a-2 and a-3, b-2 and b-3, c-2 and c-3, d-2 and d-3 and e-2 and e-3 merging.
Fig. 3 is that flow cytometry quantitative analysis triple helical beta-glucan molecular weight is to beta-glucan/poly (dA)
50-DNA1 mixture genophore transfection efficiency affect result figure; Wherein, the positive control group of a in A figure (positive controls is not add any material in cell, and the transfection efficiency recording by flow cytometer is equivalent to the standard of a tolerance), b is naked DNA 1 – poly (dA)
50the positive rate of sequence, c, d, e and f are respectively DNA1 – poly (dA)
50/ s-LNT1, DNA1 – poly (dA)
50/ s-LNT2, DNA1 – poly (dA)
50/ s-LNT3 and DNA1 – poly (dA)
50the positive rate of/s-LNT4.
Fig. 4 is different molecular weight triple helical beta-glucan/poly (dA)
nthe particle size analysis of-DNA1 mixture genophore and potentiometric analysis result figure; Wherein, a: particle size analysis result, b: potentiometric analysis result.
Fig. 5 is immune factor IL-6 and IL-12p40 burst size qualitative analysis Poly (dA)
nlength is to beta-glucan/poly (dA)
n-DNA2 mixture genophore transfection efficiency affect result figure; A:IL-12p40, b:IL-6.
Fig. 6 is flow cytometry quantitative analysis Poly (dA)
nlength is to beta-glucan/poly (dA)
n-DNA2 mixture genophore transfection efficiency affect result figure; Wherein, the positive control group of a, b1 and b2 are respectively DNA1 – poly (dA)
15, DNA1 – poly (dA)
15the positive rate of/s-LNT2 sequence; C1 and c2 are respectively DNA1 – poly (dA)
30, DNA1 – poly (dA)
30the positive rate of/s-LNT2 sequence; D1 and d2 are respectively DNA1 – poly (dA)
50, DNA1 – poly (dA)
50the positive rate of/s-LNT2 sequence.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.If do not specialize, biochemical reagents used in embodiment are commercially available analytical reagent, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Mushroom triple helical beta-glucan (t-LNT) used in following embodiment produces mushroom fruiting body and extracts from Fujian, and extracting method refers to triple helical lentinan and method for making and the purposes that mono-kind of ZL 200410060850.7(has anti-tumor activity).In order to obtain the polysaccharide sample compared with narrow molecular weight distributions, the polysaccharide of extraction is dissolved in ultrapure water again, then, at room temperature in the aqueous solution of polysaccharide, dropwise add acetone, until solution becomes muddy.The mixture of gained is warming up to 50 ℃ and keeps 2 hours, then standing over night at room temperature.By mixture with 8000rpm centrifugal 30 minutes, with separation of supernatant and gel phase, and gel phase is dissolved in ultrapure water again subsequently, distill water dialysis 5 days, filters, and lyophilize, obtains blank sheet shape sample.In order to obtain the t-LNT sample of different molecular weight, be dissolved in ultrapure water, then ultrasonic degradation different time under cell crushing instrument 400-600W.The weight-average molecular weight of t-LNT (Mw) is measured by multi-angle laser light scattering and size exclusion chromatograph joint used (Wyatt DAWN DSP, Santa Barbara, CA).
Embodiment 1
(1) 0.1g mushroom triple helical beta-glucan (being labeled as t-LNT) is dissolved in to the abundant strand polysaccharide soln (being labeled as s-LNT) that makes sex change that dissolves in 1mL dimethyl sulfoxide solvent.
(2) by the poly-deoxyadenylic acid (poly (dA) of 5OD
n) by covalent linkage and 5OD target dna keyed jointing (purchase of Ke Cong company), be then dissolved in the phosphate buffered saline buffer of 0.01M, pH 7.4, being prepared into final concentration is the DNA solution of 1 μ g/ μ L.
(3) by 4 μ L(2) poly (dA) that obtains
n-DNA/ buffered soln joins the phosphate buffered saline buffer of 194 μ L 0.01M, pH 7.4, and then by 2 μ L(1) the sex change strand polysaccharide soln that obtains joins in solution, mixes at latter 6 ℃ standing 48 hours, can obtain mixture genophore.
In the present embodiment, t-LNT weight-average molecular weight is respectively 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).Poly-deoxyadenylic acid is respectively the poly-deoxynucleotide poly (dA) containing 15,30 and 50 deoxyadenine bases
15, poly (dA)
30, and poly (dA)
50; Target dna is the DNA1(5 '-CATTCCGAGTGTCCA-3 ' of Cy3 fluorophore mark, 15 bases), Cy3 fluorophore mark is for observing transfection approach and the cellular uptake rate with quantitative analysis target dna; After poly-deoxyadenylic acid and target dna keyed jointing, be 5 '-Cy3-DNA1 – poly (dA)
n-3 ', n is 15,30 or 50.
The mixture genophore obtaining is reprinted to 0.7%(w/v) carry out electrophoresis test in sepharose, result (Fig. 1) demonstration, sex change mushroom triple helical beta-glucan and the Cy3-DNA1 – poly (dA) 50 of different molecular weight can form mixture genophore; The molecular weight of choosing is that 2.1 * 105 mushroom triple helical beta-glucans also can form mixture genophore from the nucleotide sequence with different poly-deoxyadenylic acid sequence lengths, but stable composite strengthens with the increase of poly-deoxyadenylic acid sequence length, learn that thus poly-deoxyadenylic acid sequence length is also the key factor that builds genophore, build genophore, deoxyadenine base number is at least greater than 15.
(Nikon C1-si TE 2000, Japan) for qualitative observation with analyze the target dna sequence of Cy3 fluorophor mark in the transfection behavior of RAW 264.7 cells for Laser Scanning Confocal Microscope.By cell, in culture dish, (diameter is 20 mm) cultivates after 24 hours, with aseptic PBS(0.01M, pH 7.4) washed cell 2 times, by above-mentioned 200 μ L Cy3-DNA1 – SS – poly (dA)
50/ s-LNT solid support material (being the mixture genophore that the present embodiment obtains) adds in 2mL fresh culture, with co-culture of cells 48 hours, abandons substratum, by PBS washing for cell 3 times.Nucleus Hoechst 33258(10%, v/v) dye 20 minutes, again, with after PBS washing 3 times, carry out confocal fluorescent detection.
The uptake ratio flow cytometric analysis of cell to solid support material.By RAW 264.7 cells with 1.0 * 10
5/ hole is inoculated in 6 orifice plates, adds 2mL DMEM culture medium culturing after 24 hours, changes fresh culture, and in each hole, adds 200 μ L solid support materials.Hatch after 48 hours, use PBS washed cell 3 times.With trypsin digestion and cell, and with centrifugal 5 minutes collecting cells of 1000 rpm.Abandon supernatant liquor, use twice of PBS washed cell.After finally cell suspending liquid being filtered with nylon membrane, with flow cytometer, detect (BD FACSAria III, USA).
The particle diameter of four kinds of mixture genophores and zeta current potential use particle diameter potentiometric analyzer (Nano-ZS ZEN3600, Malvern Instruments, UK) 25 ℃ of measurements.
Above-mentioned experimental result shows: molecular weight is 2.1 * 10
5the carrier that mushroom triple helical beta-glucan preparation forms has the highest transfection efficiency (Fig. 2 and 3), the excessive or too small expression that is all unfavorable for gene transfection of molecular weight, and mixture genophore shows electronegativity (Fig. 4) simultaneously.
Embodiment 2
(1) by 0.01g molecular weight, be 2.1 * 10
5mushroom triple helical beta-glucan be dissolved in the phosphate buffered saline buffer (0.01M, pH 7.4) of 1mL and fully and dissolve, then in high-temperature high-voltage reaction pipe, at 140 ℃, heat the strand polysaccharide soln that makes sex change for 30 minutes.
(2) by the poly-deoxyadenylic acid (poly (dA) of 5OD
n) by covalent linkage and 5OD target dna keyed jointing (buying from company), be then dissolved in 0.01M, pH 7.4 phosphate buffered saline buffers, being prepared into final concentration is the DNA solution of 1 μ g/ μ L.Wherein, poly (dA)
nbe respectively poly (dA)
15, poly (dA)
30, and poly (dA)
50; Target dna (DNA2) is functional non-oligodeoxynucleoside phosphorothioate that methylates (CpG DNA:1826,5 '-TCCATGACGTTCCTGACGTT-3 ', 20 bases), in order to investigate DNA transfection success or not; Meanwhile, CpG DNA transfection is entered in body, for Gene immunotherapy provides scientific basis.
(3) by 16 μ L(2) poly (dA) that obtains
n-DNA2 solution joins 16 μ L(1) in the sex change strand polysaccharide soln that obtains, mix at latter 37 ℃ standing 12 hours, obtain mixture genophore.
(4) RAW 264.7 cells are inoculated in 96 orifice plates (4.0 * 10
5cells/well), in, make cell attachment 12 hours.Until cell culture, after 12 hours, with phosphate buffer wash cell, change fresh culture, and add s-LNT2 (8 μ g/ μ L), DNA2 – poly (dA)
15, DNA2 – poly (dA)
15/ s-LNT2, DNA2 – poly (dA)
30, DNA2 – poly (dA)
30/ s-LNT2, DNA2 – poly (dA)
50and DNA2 – poly (dA)
50in/s-LNT2(all material, contain 0.8 μ g/ μ L DNA2 and 8 μ g/ μ L s-LNT2) cultivate, after 24 hours, collect substratum high speed centrifugation, and with ELISA test kit (Mouse IL-6 precoated ELISA kit and Mouse IL-12p40 precoated ELISA kit) (reaching section is, China), detect the content of cytokine IL-6 and IL-12p40 in supernatant liquor.Detecting the concrete operations specification sheets that the concrete operations of cytokine give in can reference reagent box detects.
The uptake ratio flow cytometric analysis of cell to solid support material.By RAW 264.7 cells with 1.0 * 10
5/ hole is inoculated in 6 orifice plates, adds 2mL DMEM culture medium culturing after 24 hours, changes fresh culture, and in each hole, adds 200 μ L solid support materials.Hatch after 48 hours, use PBS washed cell 3 times.With trypsin digestion and cell, and with centrifugal 5 minutes collecting cells of 100rpm.Abandon supernatant liquor, use twice of PBS washed cell.After finally cell suspending liquid being filtered with nylon membrane, with flow cytometer, detect (BD FACSAria III, USA).
Result shows, weight-average molecular weight is 2.1 * 10
5mushroom triple helical beta-glucan and DNA2 – poly (dA)
50the carrier that compound preparation forms can carry out genetic expression well, and Promote immunity factor IL-6 and IL-12p40 secrete (Fig. 5,6) largely, can be for Gene immunotherapy.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan University
<120> beta-glucan genophore and its preparation method and application
<130> 1
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA1
<400> 1
cattccgagt gtcca 15
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA2
<400> 2
tccatgacgt tcctgacgtt 20
Claims (10)
1. a preparation method for beta-glucan genophore, is characterized in that comprising the steps:
(1) beta-glucan is dissolved in phosphate buffered saline buffer, at 130-145 ℃, heat 15-60 minute be dissolved in dimethyl sulfoxide (DMSO) or strong base solution in make the strand polysaccharide soln of sex change;
(2) by poly (dA)
nby covalent linkage and target Nucleotide keyed jointing and be dissolved in pH 7-9 damping fluid;
(3) the strand polysaccharide soln of the sex change (1) being obtained joins the poly (dA) that (2) obtain
nin-target Nucleotide/buffered soln, mix, standing 12-48 hour, obtains beta-glucan genophore.
2. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: the beta-glucan described in step (1) is triple helical beta-glucan, and its molecular weight is 5.0 * 10
4-4.5 * 10
5.
3. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: the concentration > 0.1M of the strong base solution described in step (1).
4. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: the poly (dA) described in step (2)
nmiddle n>=15.
5. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: the target Nucleotide described in step (2) comprises DNA and RNA.
6. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: final concentration >=8 of the final concentration/nucleic acid of sex change strand polysaccharide in step (3).
7. the preparation method of beta-glucan genophore according to claim 1, is characterized in that: in step (3), standing temperature is 4-37 ℃.
8. a beta-glucan genophore, is characterized in that: by the method described in claim 1-7 any one, prepare.
9. the application of beta-glucan genophore claimed in claim 8 in the reprinting of DNA and RNA.
10. application according to claim 9, is characterized in that: the application of beta-glucan genophore claimed in claim 8 in the reprinting of CpG DNA and siRNA.
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| WO2019223699A1 (en) * | 2018-05-23 | 2019-11-28 | 浙江立恩生物科技有限公司 | Biological polysaccharide for preventing and treating acne and steroid-dependent dermatitis and use thereof |
| WO2020233681A1 (en) * | 2019-05-21 | 2020-11-26 | 浙江立恩生物科技有限公司 | Biological polysaccharide having effect of preventing and treating hormone-dependent dermatitis and application thereof |
| CN113045682A (en) * | 2021-03-11 | 2021-06-29 | 内蒙古大学 | Cationic polysaccharide and preparation method and application thereof |
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| CN1613876A (en) * | 2004-09-14 | 2005-05-11 | 武汉大学 | Three spiral lentinan with anti-cancer activity and its preparation and use |
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| 刘青业等: "聚脱氧腺嘌呤核苷酸与香菇-葡聚糖通过二硫键构建基因转染体系", 《2013年全国高分子学术论文报告会》, 16 October 2013 (2013-10-16) * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019223699A1 (en) * | 2018-05-23 | 2019-11-28 | 浙江立恩生物科技有限公司 | Biological polysaccharide for preventing and treating acne and steroid-dependent dermatitis and use thereof |
| CN110522761A (en) * | 2018-05-23 | 2019-12-03 | 浙江立恩生物科技有限公司 | It is a kind of to have effects that prevent and treat the biological polyoses of acne and its application |
| WO2020233681A1 (en) * | 2019-05-21 | 2020-11-26 | 浙江立恩生物科技有限公司 | Biological polysaccharide having effect of preventing and treating hormone-dependent dermatitis and application thereof |
| CN113045682A (en) * | 2021-03-11 | 2021-06-29 | 内蒙古大学 | Cationic polysaccharide and preparation method and application thereof |
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