CN104758950A - Preparation method of plasmid transmission carrier - Google Patents
Preparation method of plasmid transmission carrier Download PDFInfo
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- CN104758950A CN104758950A CN201510045315.2A CN201510045315A CN104758950A CN 104758950 A CN104758950 A CN 104758950A CN 201510045315 A CN201510045315 A CN 201510045315A CN 104758950 A CN104758950 A CN 104758950A
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- plasmid
- meerschaum
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- montmorillonitum
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Abstract
The invention discloses a plasmid DNA molecule transmission carrier. The transmission carrier contains an active component which comprises meerschaum-montmorillonite-diatomite mixed nanometer particles. The plasmid DNA molecule transmission carrier has a small particle size, good adsorption capability, good plasmid compounding capability, low cytotoxicity and an effectively improved conversion rate, is a safe and efficient plasmid transmission carrier and has a good application prospect in a plasmid transmission conversion kit.
Description
Technical field
The present invention relates to biomedical sector, be specifically related to a kind of preparation method that plasmid can be adsorbed in the transmission carrier on surface.
Background technology
Plasmid is a kind of little double-stranded DNA ring molecule naturally existed in antibacterial and yeast, and they copy with independently unit.Although only occupy the sub-fraction of host cell STb gene, with important gene.Just because plasmid DNA is less, and with resistant gene, make it to be widely used in recombinant DNA technology.
Various countries scientist is perplexed in the research of plasmid DNA transport agent always, becomes the key issue of gene therapy success or failure.People are devoted to find suitable carrier material always can be transported to target cell by plasmid DNA effectively, and maintains its effect within a certain period of time.Along with developing by leaps and bounds of nanometer biotechnology, nano-gene carrier, as a kind of non-viral-based gene carrier, although transfection efficiency is lower than viral vector, but has that immunogenicity is low, low toxicity, struck capacity is large, prepare the advantages such as simple.The system of presenting of nano-particle has the potential of targeted delivery, has good stability and biocompatibility, and energy available protecting bioactive molecule to the toleration of physiological environment, and strengthens cell to the absorption being passed molecule.It is worthy of note that nano-particle passes through increase infiltration and retention effect (EPR effect) can promote that medicine is in the enrichment of tumor tissues especially, and effectively by tumor locus Cell uptake, Cytoplasm and various organelle can be entered.In addition; there is the degradable nano-gene carrier energy available protecting plasmid DNA molecule of good biocompatibility; plasmid DNA is discharged rapidly in Cytoplasm, and realize the object that blocking gene is expressed, this will make this kind of carrier have better advantage and application prospect in plasmid DNA administration.Non-virus nano based on nano-particle transmits carrier and can be divided into the preparation of use organic material and use inorganic material preparation.Modal at present have nanotrees, Polyisobutyl cyanoacrylate and polylactic acid, alkanisation nano silicon particles etc.By these carrier molecules and the mixed complex obtaining the two mutually of the solution containing plasmid, thus realize presenting the object that plasmid DNA reaches therapeutic effect.But these class methods not easily amplify scale and repeatability is poor.
Meerschaum (sepiolite) is a kind of rich fibrous magnesium silicate clay mineral, belongs to extraordinary rare nonmetallic ore, has the title of " soft gold ", has silky luster, sometimes in wax-like gloss, and Mohs' hardness 2-2.5 level, density 1-2.2g/cm
3, plasticity is good, dissolves in hydrochloric acid.Because meerschaum has special tectonic, make it maintain a series of brilliant road, thus have great specific surface area, by meerschaum crystal structure model, calculate theoretically, the external surface area of meerschaum can reach 400m
2/ g, inner ratio surface area can reach 500m
2/ g, therefore have extremely strong adsorptivity, decolourising property and dispersibility.The basic structural formula of meerschaum belongs to chain structure, the open type ditch pivot formed in structure and crystal longer axis parallel, and thus the absorption property of this ditch pivot is extremely strong, and this is also that meerschaum becomes in these race's mineral the key point with optimum performance and most extensive use.Meerschaum is also widely used in every field because of the unique physico-chemical character had.Meerschaum can realize plasmid to transformation; This medium is friendly to health, without carcinogen; Itself inactive, can not have an impact to host growth; Special equipment is not needed in operating process; Do not need competent preparation just can obtain higher conversion ratio.But generally, meerschaum head grade is low, and impurity component is numerous and diverse and unstable, and purifying technique is relatively complicated.
Montmorillonitum (MMT) be bentonite again, is a kind of mineral drug.China's Montmorillonitum is medicinal with a long history according to records, and the dry ground in the Tang Dynasty medical science masterpiece supplement to the Herbal is Montmorillonitum.Domestic Montmorillonitum aboundresources, starts to be manufactured pharmaceutical excipient by American-European countries from the eighties in 20th century, and includes the U.S., Europe and British Pharmacopoeia respectively in.The insoluble phyllosilicate solid matter that Montmorillonitum system is made up of silicon oxide and aluminium oxide, due to the internal structure that it is special, produce transducing power with undersaturated negative charge and the strong cation of tool, have absolute absorption fixation to the virus and bacteria and DNA that enter human body.Nanometer Montmorillonitum significantly improves relative to the former stone specific surface area of Montmorillonitum, and absorbability significantly strengthens; Utilize the avirulence of nanometer Montmorillonitum, high-specific surface area, high adsorption, to characteristics such as the adsorption effects of plasmid.But in alkaline environment, the adsorbance of Montmorillonitum to DNA significantly decreases, so its acid-base value to whole system requires harsher.
Kieselguhr is a kind of biogenic silicastone, primarily of ancient times algae and the siliceous remains of a part of Radiolaria formed.Kieselguhr has porous, low-density, the features such as large specific surface area, and also has the special nature such as relative Incoercibility and chemical stability.Diatomite nano grade particles has very large surface energy at high chaotropic agent, also has larger elecrtonegativity, and in conjunction with large quantity of moisture in low salt solutions, DNA is equally in conjunction with large quantity of moisture.After DNA is combined with kieselguhr, diatomite nano grade particles well protects DNA to shear from the degraded of enzyme and other.But the ability of diatomite adsorption DNA has significant change with the granular size of itself, excessively thick particle DNA absorbability is extremely weak, should not apply.
Domestic and international at present above three kinds of materials to be had carried out some research in the application of environment and biological field, but for molecular biology and pharmaceutical field research less.It is not yet seen three kinds of nano-particle in conjunction with respective pluses and minuses coupling transmit the report of carrier as plasmid DNA molecule.
Summary of the invention
Of the present invention solved technical problem is to provide a kind of plasmid DNA to transmit carrier, it has, and particle diameter is little, the character of high adsorption capacity, having good plasmid DNA compound ability, low cytotoxicity and cell endocytic advantage, is that a safety, efficiently plasmid DNA transmit carrier.
The object of the invention is to provide a kind of transmission carrier that plasmid DNA can be adsorbed in surface, and its active component is meerschaum, Montmorillonitum and diatomaceous mixing nano-particle.The adsorbable plasmid DNA molecule of this transmission carrier, wherein, the mean diameter of described transmission carrier nanoparticles is 25nm.
Described plasmid DNA transport agent can not change or reduce the effect of plasmid DNA in subsequent process.Wherein, the transmission carrier of described plasmid DNA is in the exploitation purposes of related kit and the application in related drugs.
Detailed description of the invention
Embodiment 1: the preparation of meerschaum nano material
10g meerschaum original soil ore deposit is placed in 100mL distilled water and soaks 24h, after its immersion is loose, adds 0.1g tetrasodium pyrophosphate, stir 2h with blender, staticly settle.After there are the stable beds of precipitation, the HCl regulation system pH adding 0.5mol/L is 6.0, fully stirs 20min, suspension is above poured out, uses vacuum pump sucking filtration, then with distilled water wash for several times, then filter cake is put into baking oven to dry, grind, after autoclaving, sealing saves backup.
Embodiment 2: the preparation of nano-montmorillonite
Adding 6. 0g Montmorillonitums in 100mL distilled water, using magnetic stirrer to dissolving completely.Rotating speed is 5000r/min, centrifugalize 2h.Precipitation is proceeded to evaporating dish, in 65 DEG C of vacuum drying 6-10h, is ground to 200 orders, then in 65 DEG C of vacuum drying 2-4h, after autoclaving, sealing saves backup.
Embodiment 3: diatomaceous preparation
Take 10g kieselguhr, suspend with 50mL distilled water and pour into gently and be equipped with in the Falcone pipe of 450mL distilled water, sonic oscillation 10min, reclaim suspension after leaving standstill 5min, suspension through the centrifugal 30s of 12000rpm, abandons supernatant again.The granule equal-volume distilled water of centrifugal gained suspends, and uses vacuum pump sucking filtration, then with distilled water wash several, then filter cake is put into baking oven and dry, and grinds.After autoclaving, 4 DEG C save backup.
Embodiment 4: the preparation of mixing nano material
Under the prerequisite of fixed temperature (room temperature), mixing time (12h), system pH (7.4) and mixing speed (400r/min), for probe into meerschaum, Montmorillonitum, diatomite nano material the change of amount ratio on the impact of the conversion ratio of recombinant plasmid vector, we be used alone meerschaum and be provided with meerschaum, Montmorillonitum, the score of kieselguhr quality do not carry out subsequent transformation experiment for 1:0:1,1:1:0,2:1:1,1:1:1,1:1:2.Concrete operations are: take meerschaum, Montmorillonitum, kieselguhr respectively according to the above ratio with electronic balance, are resuspended in 5ml distilled water, ultrasonic disperse 15min; Centrifugal, abandon supernatant, precipitation is resuspended in 5mL containing in 200mmol/L KCl, 5mmol/L HEPES buffer, ultrasonic disperse 20min, stirring at room temperature 12h; Centrifugal again, abandon supernatant, precipitation is resuspended in appropriate distilled water, and adjust pH to 7.4, the degerming 4 DEG C of storages of film are for subsequent use excessively.
The preparation of embodiment 5:pET28a-HBc prokaryotic expression carrier
By transferring hepatitis B virus core antigen (HBc) sequence in GenBank data base, give preferences in conjunction with e. coli codon to be optimized it, obtain the complete genome sequence of HBc, the HBc complete sequence of required synthesis is analyzed, according to the result of gene sequencing, entrust Dalian Takara company to carry out full genome synthesis, and add restriction enzyme site respectively at the two ends of sequence
noci and
hind III, the aim sequence obtained is connected on pET28a carrier.
Plasmid is entrusted the restructuring gram of third party biotech firm sequence verification fall middle gene order whether with require to conform to.Result display sequencing result and aim sequence all bases of comparing all consistent with aim sequence, prove successfully to build prokaryotic expression carrier.
Embodiment 6: colibacillary conversion and abduction delivering
After mixing nano material mixes with the expression plasmid pET28a-HBc prokaryotic expression carrier built, transform and express in host e. coli BL21, even spread LB culture medium flat plate (blocking that resistance), finds after incubated overnight, being used alone meerschaum can reach 1 × 10
4the Plastid transformation rate of/μ g, and the conversion ratio that meerschaum, Montmorillonitum, kieselguhr mass ratio are 1:0:1,1:1:0,2:1:1,1:1:1,1:1:2 is respectively: 1.42 × 10
4/ μ g, 1.61 × 10
4/ μ g, 1.73 × 10
5/ μ g, 1.06 × 10
6/ μ g, 1.08 × 10
5/ μ g.Can show that meerschaum, Montmorillonitum, kieselguhr mass ratio are that the changing effect of 1:1:1 is best by result, again IPTG abduction delivering is carried out to it, take out 1mL culture as contrast before induction, in residue culture, add IPTG, make final concentration be 1mmol/L, continue to cultivate.Respectively induction 1,3,5h time sampling 1mL, induction terminates.Meanwhile, the BL21 escherichia coli transformed according to above-mentioned same method abduction delivering pET28a empty carrier, as negative control.By the abduction delivering product of different induction time centrifugal 1min under 12000r/min, then ultrasonic 3s under 300W, interval 3s, to escherichia coli ultrasonication 10min.Under 12000r/min, centrifugal 2min, gets supernatant, first adopts BCA RNA isolation kit detection by quantitative protein concentration to determine SDS-PAGE applied sample amount.Get after 20 μ L protein samples mix with 5 times of sample-loading buffers, boiling water bath 5min, loading after cooling completely, carries out SDS-PAGE (5% concentrated glue, 12% separation gel) electrophoresis, 80V constant voltage electrophoresis 3h, the abduction delivering situation of analyzing proteins.Experimental result shows, and target protein actual size is 24.2KDa, and predicts the outcome consistent, shows albumen successful expression, illustrates that nanometer hybrid particles of the present invention not only effectively can transform foreign DNA, and do not affect the abduction delivering effect of recipient E. coli.
Embodiment 7: Western blotting detects protein expression
After SDS-PAGE electrophoresis, electrotransfer is on pvdf membrane, then 2h is closed by confining liquid room temperature, spend the night with mouse-anti people HBc monoclonal antibody (1:200) 4 DEG C after rinsing, with after TBST eluting with sheep anti-mouse igg (1:10000) the incubated at room 1h of horseradish peroxidase-labeled, again with the development of ECL chemical illuminating reagent, exposure, detect the expression of HBc albumen.Western blot testing result shows, and has specific band to occur in corresponding position.After showing that nanometer hybrid particles of the present invention transforms exogenous plasmid dna, the antigenicity of its plasmid-encoded protein can not be affected.
Claims (5)
1. plasmid transmits a preparation method for carrier, and wherein said carrier contains meerschaum, Montmorillonitum and diatomaceous mixing nano-particle, and the method comprises the following steps:
First plasmid DNA is made to be dissolved in solution,
Then meerschaum, Montmorillonitum and diatomaceous nano material is prepared respectively,
Then meerschaum, Montmorillonitum and kieselguhr are prepared into mixing nano-particle in proportion,
Then plasmid DNA solution is added in mixing nano-particle, form plasmid and mixture of nanoparticles.
2. plasmid according to claim 1 transmits the preparation method of carrier, it is characterized in that the mean diameter of described transmission carrier nanoparticles is 25nm.
3. plasmid according to claim 1 and 2 transmits carrier, it is characterized in that the meerschaum of described nano-particle, Montmorillonitum and diatomaceous mixing best in quality are 1:1:1.
4. the meerschaum comprising nano-particle, Montmorillonitum and kieselguhr according to any one of claim 1-3 are preparing the purposes in test kit.
5. carried out adsorbing and the carrier transmitted by plasmid DNA molecule, it is characterized in that, described medicine contains the meerschaum of the mixing nano-particle comprised described in any one of claims 1-3, Montmorillonitum and the diatomaceous and acceptable adjuvant of pharmacy.
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| CN201510045315.2A CN104758950B (en) | 2015-01-29 | 2015-01-29 | A kind of preparation of plasmid transmitting carrier |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004065237A (en) * | 2002-06-12 | 2004-03-04 | Osaka Industrial Promotion Organization | Carrier for introducing gene, agent for introducing the gene using the carrier, method for introducing the gene using the agent, and gene-introduced cell by the method |
| US20080182786A1 (en) * | 2005-02-09 | 2008-07-31 | Meiji Dairies Corporation | Support For Protein Transfer, Protein Transfer Agent Using the Support, Protein Transfer Method, Cell Having Protein Transferred Thereinto and Method of Producing the Same |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004065237A (en) * | 2002-06-12 | 2004-03-04 | Osaka Industrial Promotion Organization | Carrier for introducing gene, agent for introducing the gene using the carrier, method for introducing the gene using the agent, and gene-introduced cell by the method |
| US20080182786A1 (en) * | 2005-02-09 | 2008-07-31 | Meiji Dairies Corporation | Support For Protein Transfer, Protein Transfer Agent Using the Support, Protein Transfer Method, Cell Having Protein Transferred Thereinto and Method of Producing the Same |
Non-Patent Citations (3)
| Title |
|---|
| ILARIA REA ET AL: "Diatomite biosilica nanocarriers for siRNA transport inside cancer cells", 《BIOCHIMICA ET BIOPHYSICA ACTA》 * |
| 夏嵩, 张成武: "硅藻纳米技术", 《中国生物工程杂志》 * |
| 李景鹏等: "三种质粒DNA对不同大肠杆菌菌株转化率的比较", 《东北农业大学学报》 * |
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