CN105727310B - Based on chitosan package ALV-J P-miRNA-env recombinant plasmid nano-complex and its preparation method and application - Google Patents

Based on chitosan package ALV-J P-miRNA-env recombinant plasmid nano-complex and its preparation method and application Download PDF

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CN105727310B
CN105727310B CN201610068971.9A CN201610068971A CN105727310B CN 105727310 B CN105727310 B CN 105727310B CN 201610068971 A CN201610068971 A CN 201610068971A CN 105727310 B CN105727310 B CN 105727310B
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CN105727310A (en
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成子强
贾雪莲
张利
崔熙尧
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Shandong Agricultural University
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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Abstract

The present invention relates to bio-pharmaceuticals and field of virology, specifically provide the technology of preparing and application of a kind of small interference genomic medicine, more specifically it is related to based on natural cationic carrier chitosan and J- subgroup avian leucosis ALV-JP-miRNA-env gene shape nano-complex and its preparation method and application, the effect using CTS-P-miRNA-env nano-complex prepared by the present invention than the inhibition ALV-J virus replication of former gymnoplasm grain becomes apparent from as the result is shown, J subgroup avian leucosis can be more effectively prevented and treated to occur and propagate, new antiviral method and means are provided for avian leukosis prevention and control, also technical support is provided at the clinical application of anti-avian leukosis biological agent for it.

Description

Based on chitosan wrap up ALV-J P-miRNA-env recombinant plasmid nano-complex and Preparation method and application
(1) technical field
The present invention relates to biological medicines and field of virology, and in particular to a kind of ALV-J P- based on chitosan package MiRNA-env recombinant plasmid nano-complex and its preparation method and application.
(2) background technique
J subgroup avian leucosis is a kind of tumor disease as caused by J subgroup avian leucosis virus (ALV-J), main Fowl clinical manifestation is myelocytic leukemia, and is mostly swollen with immune tolerance, high mortality, growth retardation and more histoorgans Tumor etc. is main feature, and at ascendant trend, clinical case has developed to commodity from commercial broiler group for the generation of China ALV-J Laying hen, local breeder flock and other poultry, occurrence characteristic mainly contain morbidity early, infection rate and disease incidence be high, host extensively, Pathogenic strong, tumour diversification etc..In addition, the fowl tumor disease based on ALV-J can also be with Marek's disease, reticular endothelium The tumor diseases mixed infection such as hyperplasia disease, this greatly increases the prevention and treatment difficulty of disease, brings to aviculture huge Economic loss.
Due to the hypervariability and the immune evasion ability of its own of ALV-J viral envelope proteins, so far to the prevention and treatment of ALV-J Research yet there are no remarkable effect, and there is no the drug or vaccine for effectively controlling and preventing and treating, and also not truly feasible prevention and control are arranged It applies.Foreign countries be the occurrence of this disease is controlled by purifying, but its period it is long, it is at high cost etc. due to, the country also fails at present Effectively implement, jumpbogroup purification can only be carried out by eliminating infected chicken.In addition, unreasonable, unscientific control techniques, can also accelerate The evolution and mutation of virus, make disease more sophisticated, the aquiculture status based on China seeks new technology, the new hand of ALV-J prevention and control Section has practical significance.
In recent years, with the continuous development of Protocols in Molecular Biology and genetically engineered drug, to genomic medicine and its delivering Systematic research becomes the hot spot of current research, and chitosan is unique alkaline polysaccharide in nature, not soluble in water, dissolves in vinegar The dilute acid solns such as acid and inorganic acid.It is by linear polymer chitin through de- second phthalein reaction its chemical name is Chitosan Product afterwards.Chitin is a kind of natural polymer, and in nature, chitin is widely present in unicellular lower eukaryote mushroom, The cell of algae saves the shell of branch animal shrimp, crab, insect, the inner casing and cartilage of mollusk (such as squid, bird thief), high plant The cell wall etc. of object, the annual life synthesis resource of chitin are second that plant fiber is only second on the earth up to 200,000,000,000 tons Big living resources are the inexhaustible living resources of the mankind.N- second phthalidyl in chitin structural formula on glycosyl is largely gone It is exactly mostly important derivative -- the chitosan of chitin if falling, deacetylation is for characterizing chitosan macromolecular chain Middle primary amino group content, chitosan of the deacetylation greater than 85% is known as chitosan with high deacetylation degree, contains a large amount of primary amino group. Primary amino group is positively charged in slant acidity condition, can adsorption of DNA or RNA well, and primary amino group has binding hydrogen ions Ability, therefore chitosan has certain buffer capacity, and chitosan can be helped to flee from interior corpusculum.It is from a wealth of sources, has good Good biocompatibility and biological degradability, it is non-toxic to humans；Chitosan is used as in the prior art corresponds to as carrier carrying Tiny RNA or the technical solutions of other targeted drugs related in the tumour medicine of people's medicine, nucleic acid drug field, but in fowl It always is blank in terms of quasi-leukemia, how to obtain anti-avian leukosis biological agent using chitosan and is asked as urgently to be resolved One of topic.
(3) summary of the invention
For the existing above problem, the present invention provides a kind of technology of preparing of small interference genomic medicine and answer With being more specifically related to based on natural cationic carrier chitosan and J- subgroup avian leucosis ALV-JP-miRNA-env gene Shape nano-complex and its preparation method and application, it is multiple using CTS-P-miRNA-env nanometers prepared by the present invention as the result is shown The effect that object is closed than the inhibition ALV-J virus replication of former gymnoplasm grain becomes apparent from, and can more effectively prevent and treat J subgroup avian leucosis Occur and propagate, new antiviral method and means are provided for avian leukosis prevention and control, is also it into anti-avian leukosis biology system The clinical application of agent provides technical support.
The present invention is that the further investigation carried out based on following theoretical basis is obtained:
Small interference genomic medicine is a kind of current biological medicine technology state-of-the-art in the world, either domestic or state Border is establishing more targeted drug design reinforcement small RNA curative effect of medication and is improving the innovative technology of internal drug importing efficiency Upper development is with rapid changepl. never-ending changes and improvements.
RNA interference (RNAi) is a kind of biological phenomena generally existing in vivo of discovered in recent years.Utilize RNA Perturbation technique can specificity inhibit Disease-causing gene expression, have efficiently and diversified feature, in the poultry including tumour Great potential in avian disease treatment.
In RNAi drug development, on the one hand, it needs to constantly discover new target spot and understands fully its function and mechanism of action, from And the RNA drug for instructing clinical development effective is for bringing real Gospel for culturist in major disease diagnosing and treating. On the other hand, since siRNA stability is poor, it is easy to degrade, cellular uptake is poor, transports into cytoplasm low efficiency and lacks targeting energy Power, therefore another important key of small interference genomic medicine exploitation is the efficient delivery system of exploitation.
Chitosan surface has positive charge, passes through electrostatic adsorption and carries negatively charged recombinant plasmid base Cause, to prevent degradation of the nucleic acid in vivo enzyme to Plasmid DNA by space steric effect；Chitosan is water soluble polymer, can Solve the sedimentation problem in genophore preparation and transfection process；Chitosan is polysaccharose substance, can be with certain tumor surfaces Polysaccharide receptor combines, and increases the tumor-targeting of such genophore；Chitosan is biodegradable high molecular material, can be led to The control slow release that nanometer biotechnology reaches gene is crossed, so that gene therapy be made to reach optimum efficiency.Therefore base of the present invention In the envelope protein structural gene (env) that can significantly inhibit ALV-J that screening obtains, siRNA is obtained with expression and recombinates table It is targeted drug up to plasmid, selects chitosan nanoparticle as the pharmaceutical carrier of ALV-J clinical preparation, prepare anti-ALV-J recombination table Up to interference plasmid-chitosan nano compound clinical preparation.
It is of the invention that the specific implementation steps are as follows:
Endotoxin is carried out to P-miRNA-env recombinant plasmid largely to extract, and identification is sequenced, to chitosan, P- The preparation condition of miRNA-env nano-complex is groped；To the chitosan of preparation, P-miRNA-env nano-complex into Row partial size, form, encapsulation rate, anti-DNAseI digestion power etc. are detected；Cell transfecting detects chitosan, P-miRNA-env Nano-complex inhibits ALV-J virus replication effect in vitro and explores its effective dose,
Wherein CTS-P-miRNA-env nano-complex the preparation method is as follows:
CTS-P-miR-env nano-complex is prepared using complex coacervation: by 60 P-miR-envs of the μ g without RNase (0.225ug/ μ L) is added in 1mg/ml TPP aqueous solution 1mL, is uniformly mixed；By P-miR-env-TPP solution 2ml syringe needle Point drop-wise instills in the chitosan-acetic acid solution that 3mL concentration is 2mg/ml, rate of addition 40d-50d/min, whirlpool after instillation 30s is mixed, 30min is incubated at room temperature；Collect nano-complex suspension in no RNAse, the EP pipe without DNase to obtain the final product；
By controlling above-mentioned preparation process, the finally obtained nano-complex particle size range of the present invention is in 100 rans (90-110 nanometers) there is no the product of this particle size range in the prior art, and the entrance of the partial size of this range is dynamic It is more easier in object, transfection efficiency is more preferable, also different compared with human body use scope；
In addition to this invention also includes the conversion of P-miRNA-env recombinant plasmid and extraction and chitosan blank nanometers The preparation of grain.
The nano-complex properties that the present invention obtains detect to obtain index as follows:
(1) transmission electron microscope observing: nano-complex form is uniform, and spherical in shape or spherical, size is in 100nm or so；(2) Particle size measurer analysis measurement nano-complex partial size, 98% nano-complex partial size is 103 nanometers, and dispersion degree is good；(3) The average value of the measure and calculation encapsulation rate of nano-complex encapsulation rate is 88%.The nano-complex that indices illustrate Functional, stable structure lays the foundation for next step cell transfecting.
In order to further verify the performance of the nano-complex, inventor has carried out DF-1 cell transfecting: selection cell turns Common agents liposome is contaminated as control, while cell without any processing is set as blank control, by CTS-P-miR- Basic, normal, high 3 dosage of env nano-complex point transfects cell, uses fluorescence inverted microscope respectively in 24,48,96h With flow cytomery transfection efficiency and transfection efficiency；Cytotoxicity is detected with MTT, Real-time PCR detects group of cells The expression of middle ALV-J mRNA；Western blot detects viral envelope proteins expression.
The results show that CTS-P-miR-env nano-complex is low, middle dose group and high dose group transfection efficiency are above lipid Body control group, and cytotoxicity is lower than control liposome group.Real-time PCR detects CTS-P-miR-env nano-complex Better inhibitory effect is played to the breeding ratio gymnoplasm grain of ALV-J, Western blot detects the expression of ALV-J envelope protein Amount illustrates that the CTS-P-miR-env being prepared receives compared with having apparent reduction for ALV-J control liposome group and gymnoplasm grain group Rice compound in terms of the duplication and expression for inhibiting ALV-J more compared with the plasmid group function and effect that gymnoplasm grain and liposome are transfection agents It is good.
In conclusion compared with prior art present invention determine that the P-miR-env based on chitosan package forms nanometer The method that compound prepares the small interference genomic medicine of anti-ALV-J clinical preparation, CTS-P-miR-env nano-complex can effectively press down The duplication of J subgroup avian leucosis virus processed provides the foundation for antiviral biological agent and clinical application, while providing optimization The preparation condition of chitosan, P-miR-env recombinant plasmid afterwards, has further determined CTS-P-miR-env nano-complex The effective dose of suppressing virus replication, and then provide a kind of method that the infection of J subgroup avian leucosis virus can be effectively controlled, solution Aviculture of having determined J subgroup avian leucosis virus infects the problem that effectively controls, is compared with the traditional method compared with can significantly reduce Directly or indirectly economic loss caused by J subgroup avian leucosis has started the new approaches of fowl tumprigenicity prevention and cure of viruses research.
(4) Detailed description of the invention
Fig. 1 is CTS-P-miR-env nano-complex grain diameter measurement figure,
As seen from the figure, it is uniform to obtain nano-complex form by the present invention, spherical in shape or spherical, size in 100nm or so, Wherein 98% nano-complex partial size is 103 rans；
Fig. 2 is that the anti-DnaseI of CTS-P-miR-env nano-complex digests electrophoretogram,
It is gymnoplasm grain that M, which is DNA Maker, 1 and 2, in figure, and 3-5 is CTS-P-miR-env nanometers obtained of the present invention multiple Object is closed,
As seen from the figure, 1 and 2 gymnoplasm grains are completely degraded, and in well and band almost disappears, and receives in 3-5 Rice compound is blocked in well completely, illustrates that CTS can effectively play Plasmid DNA in nano-complex of the invention To protection；
Fig. 3 is the transmission electron microscope picture of different visual angle nanoparticles,
It can be seen that the partial size for the nanoparticle seen at different visual angles, is all in 100 rans；
Fig. 4 is chitosan P-miR-env gel blocking electrophoretogram,
M is DNA Maker in figure, and 1 is CTS-P-miR-env blank, and 2 be gymnoplasm grain, and 3 be present invention CTS- obtained P-miR-env nano-complex,
As seen from the figure, the gymnoplasm grain of only 2 swimming lanes excludes loading wells, and 2 and 3 be to portal, it is seen that chitosan nano particle Plasmid can be effectively combined by the function of classics, retardance recombinant plasmid is mobile and is stranded in well；
Fig. 5 is CTS-P-miR-env nano-complex transfection efficiency figure；
Fig. 6 is CTS-P-miR-env nano-complex suppressing virus replication result figure.
Specific embodiment
In the present embodiment unless otherwise specified, other are all made of prior art completion.
The conversion of 1 plasmid of embodiment and endotoxin is gone to extract
The big extraction reagent kit of endotoxin plasmid is gone to carry out using Tiangeng Biotechnology Co., Ltd: (described solution P1 etc. It is all to be carried in kit)
1.1 conversion
(1) DH5 α is taken out, is placed in ice water, in super-clean bench, every Guan Zhongjia (10 μ L) recombinant plasmid dna is mixed, ice bath In put 30-40min；
(2) conversion tube is put into 42 ° of water-baths, stringent timing 90s；
(3) pipe is transferred on ice rapidly after, places 2-3min；
(4) it takes to super-clean bench, 900 μ LLB fluid nutrient mediums of addition, 37 ° of oscillation 45-60min,
(5) 3000r is centrifuged 2min, discards 500 μ L, and residue is resuspended, is then coated on the plate of Spectinomycin resistance, 37 ° of cultures after forty minutes, are inverted culture 16h；
(6) next day, super-clean bench picking single colonie (first draw single bacterium colony, take 2 test tubes, every pipe adds 200ml liquid The spectinomycin of+200 μ L of LB culture medium presss from both sides most small gauge needles with tweezers and chooses bacterium, is put into test tube) it is placed in test tube, 37 ° are shaken bacterium 6-10h；
1.2P-miR-env recombinant plasmid goes endotoxin to extract
(1) column equilibration step: into adsorption column CP6, the equilibrium liquid of 2.5ml is added in (adsorption column is put into 50m1 collecting pipe) BL, 8000rPm are centrifuged 2min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe；
(2) centrifuge tube is added in the bacterium solution for taking 100ml to be incubated overnight, and room temperature 8000rPm is centrifuged 3min and absorbs supernatant collection carefully Bacterium；
(3) 8ml solution P1 is added in the centrifuge tube of Xiang Liuyou bacterial sediment, is thoroughly hanged using pipettor or turbula shaker Floating bacterium cell pellet；
(4) 8ml solution P2 is added into centrifuge tube, spins upside down 6-8 times immediately, is placed at room temperature for 5min；
(5) 8ml solution P4 is added into centrifuge tube, spins upside down 6-8 times, mixes well immediately, until white occurs in solution Disperse flocculent deposit.Then it is placed at room temperature for 10min, 8000rPm is centrifuged 5-10min, makes white precipitate to tube bottom, incites somebody to action all molten Liquid is carefully poured into filter and is filtered, and filtrate is collected in the pipe of clean 50ml；
(6) isopropanol of 0.3 times of filtrate volume is added into filtrate, the suction being transferred in collecting pipe after mixing of turning upside down In attached column CP6；
(7) room temperature 8000rPm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column CP6 is placed back in collecting pipe In；
(8) 10ml rinsing liquid PW is added into adsorption column CP6,8000rPm is centrifuged 2min, discards the waste liquid in collecting pipe, Adsorption column is placed back in collecting pipe, repeats rinsing twice；
(9) 3ml dehydrated alcohol is added into adsorption column CP6, room temperature 8000rPm is centrifuged 2min, outwells waste liquid；By adsorption column CP6 is placed back in collecting pipe, and 8000rPm is centrifuged 5min, and rinsing liquid remaining in adsorption column is removed；
(10) adsorption column CP6 is placed in a clean 50ml collecting pipe, is vacantly added dropwise to the intermediate position of adsorbed film 1-2ml elution buffer TB, is placed at room temperature for 5min, then room temperature 8, and 000rPm is centrifuged 2min, and the eluent for being centrifuged acquisition is whole A clean 1.5ml centrifuge tube is moved into, -20 DEG C of preservations obtain plasmid endotoxin-free.
The preparation method of 2 chitosan blank nanoparticle of embodiment
Chitosan blank nanoparticle is prepared using complex coacervation, by solution 2ml syringe needle in 1mL TPP (1mg/ml) solution Point drop-wise instills in 3mL chitosan (2mg/ml) acetum, and rate of addition 40d-50d/min, whirlpool mixes after instillation 30s is incubated at room temperature 30min.It collects nanoparticle suspension and both obtains chitosan blank nanometer in no RNAse, the EP pipe without DNase Grain.
The preparation of embodiment 3CTS-P-miRNA-env nano-complex
The specific method is as follows:
CTS-P-miR-env nano-complex is prepared using complex coacervation: CTS-P-miR-env is prepared using complex coacervation P-miR-env (0.225ug/ μ L) of the 60 μ g without RNase is added in 1mL TPP solution, is uniformly mixed by nanoparticle；By P- MiR-env-TPP solution is instilled in 3mL chitosan-acetic acid solution with 2ml syringe needle point drop-wise, rate of addition 40d-50d/min, Whirlpool mixing 30s after instillation is incubated at room temperature 30min.Nanoparticle suspension is collected in no RNAse, the EP pipe without DNase.
The detection of 4 nano-complex performance indexes of embodiment:
(1) transmission electron microscope observing takes the CTS-P-miR-env nanoparticle one prepared to drop to the copper mesh for attaching carbon-fiber film On, 2 minutes are stood, blots suspension with filter paper, then 2% phosphotungstic acid counterstain 5min is added dropwise, is placed in air, in throwing after drying Observed under electron microscope is penetrated, nano-complex form is uniform as the result is shown, and spherical in shape or spherical, size is in 100nm or so；
(2) in analyzer sample holes, analysis is surveyed for particle size measurer analysis plus a small amount of CTS-P-miR-env nanoparticle solution Determine nanoparticle partial size, as a result visible 98% nano-complex partial size is 103 nanometers, and dispersion degree is good.
(3) measurement of nanoparticle encapsulation rate is control with blank CTS nanoparticle, and precision measures CTS-P-miR-env nanometers Grain 0.5ml, UV spectrophotometer measuring OD260, representation DNA total amount；Take appropriate CTS-P-miR-env nanoparticle, 4 DEG C of conditions Under, the fixed centrifugation 20min of 13000 × g, precision measures supernatant 0.5ml, and 260 receive in UV spectrophotometer measuring supernatant Light absorption value at rice, this represents free amount of DNA, then is calculated by the following formula encapsulation rate: the encapsulation rate G%=[(total-W of W Trip)/W is total] × 100%, W is always the total amount of DNA in formula；The amount that W trip is the DNA to dissociate in supernatant, detects five samples, ties Fruit shows that the average value of encapsulation rate is 88%.
Embodiment 5DF-1 cell transfecting
It is divided into four groups by the difference of transfection reagent, according to illustrating for 12 orifice plate dosages:
A group is blank control group (cell does not apply any intervention)；
B group is naked P-miR-env recombinant plasmid control group；With 1.6 μ g of DNA content transfection；
C group is control liposome group (LiPofeterTMLiposome) 1.6 μ gDNA+5 μ L transfection reagents；
D group is CTS-P-miR-env nanoparticle group, is respectively 1.6 μ g with DNA content, 3.2 μ g, 6.4 μ g transfection；Every time 2 repetitions are done in transfection.
Transfection: inhaling the cell culture fluid abandoned in 12 orifice plates before transfection, the DMEM culture solution of 1ml serum-free is added, and then will The CTS-P-miR-env nano-complex or gymnoplasm grain of different volumes are added in corresponding cell hole, shake culture plate, gently It mixes.
Culture: 12 well culture plates are placed in 5% CO2In incubator, after 37 DEG C of incubation 6h, inhales and abandon transfection liquid, be changed to DMEM culture solution containing 10%FBS, continues to cultivate.
(5) it is cultivated after transfecting
Growth plate is placed in 5% CO2Incubator in, 37 DEG C of culture 24-96h, transfection is done and is repeated three times every time.
After transfection for 24 hours, the cell of 48h, 72h observation band green fluorescence under fluorescence inverted microscope, Real-time PCR Detect the expression of ALV-J mRNA in group of cells；As a result as can be seen from Figure 5 compared with the cell of untransfected plasmid, nanometer Compound transfection group transfection is better than naked plasmid control transfection group and control liposome transfection group, from Real-time PCR The expression of testing result ALV-J mRNA.
Analyze its relative value using SPSS software, as a result as shown in fig. 6, display nano-complex group and blank control group, Compared to variant significant (P < 0.05) between recessive control group, 83% is respectively as follows: to the inhibiting rate of ALV-J in DF-1 cell, 78%, the results showed that nano-complex can preferably inhibit the duplication of ALV-J, the nano-complex of preparation can more into Enter cell and carries out interference effect.
(5) specific embodiment
The technology that chitosan package P-miRNA-env recombinant plasmid forms nano-complex anti-ALV-J is prepared below to face The preparation of bed preparation is described in further detail.
1. the conversion of plasmid and endotoxin is gone to extract
Conversion
(1) DH5 α is taken out, is placed in ice water, in super-clean bench, (contained amount should be no more than in 10 μ L or volume by every Guan Zhongjia 50ng) recombinant plasmid dna mixes, and puts 30-40min in ice bath；
(2) conversion tube is put into 42 ° of water-baths, stringent timing 90s (not shake)；
(3) pipe is transferred on ice rapidly after, places 2-3min；
(4) it takes to super-clean bench, 900 μ L LB liquid mediums of addition, 37 ° of oscillation 45-60min,
(5) 3000r is centrifuged 2min, and (taking to super-clean bench) discards 500 μ L, and residue is resuspended, and it is anti-to be then coated on spectinomycin Property plate on, 37 ° culture after forty minutes, be inverted culture 16h；
(6) next day, super-clean bench picking single colonie (first draw single bacterium colony, take 2 test tubes, every pipe adds 200ml liquid The spectinomycin of+200 μ L of LB culture medium presss from both sides most small gauge needles with tweezers and chooses bacterium, is put into test tube) it is placed in test tube, 37 ° are shaken bacterium 6-10h, (muddiness)
P-miR-env recombinant plasmid goes endotoxin to mention greatly
(1) column equilibration step: into adsorption column CP6, the equilibrium liquid of 2.5ml is added in (adsorption column is put into 50m1 collecting pipe) BL, 8000rpm are centrifuged 2min, outwell the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.It (is handled with equilibrium liquid The pillar crossed preferably uses immediately).
(2) taking 100ml, (according to the suitable amount of concentration selection of culture thallus, low-copy recommendation is incubated overnight with 200ml) Bacterium solution be added centrifuge tube, room temperature 8000rpm be centrifuged 3min collect bacterium, as far as possible absorption supernatant.
Note: bacterial sediment can be collected into a centrifuge tube by being centrifuged several times when bacterium solution is more, bacterium solution amount with It can sufficiently crack and be preferred, bacterium solution, which excessively will lead to, cracks insufficient extraction efficiency to reduce plasmid.
(3) supernatant is absorbed as far as possible, and to ensure that supernatant is all drawn, the water in bottle wall is please sucked with clean blotting paper Drop.
(4) 8ml solution P1 (please first check whether and RNase A has been added) is added in the centrifuge tube of Xiang Liuyou bacterial sediment, Use pipettor or the thorough suspended bacterial cell precipitation of turbula shaker.
Note: do thoroughly suspended bacterial precipitate, if there is the fungus block not mixed thoroughly, will affect lytic effect, lead Cause extracted amount and purity relatively low.For low-copy plasmid, P1, the dosage of P2, P4 are scaling up while increasing thallus dosage.
(5) 8ml solution P2 is added into centrifuge tube, leniently spins upside down 6-8 times immediately, is placed at room temperature for 5min
Note: leniently mixing, not shake acutely, in order to avoid contaminating genomic dna.Bacterium solution should become limpid viscous at this time It is thick, may be excessive due to thallus if not becoming limpid, cracking is not thorough, and should reduce biomass.
(6) 8ml solution P4 is added into centrifuge tube, leniently spins upside down 6-8 times, mixes well immediately, until solution goes out Existing white dispersion flocculent deposit.Then it is placed at room temperature for 10min or so.8000rpm is centrifuged 5-10min, makes white precipitate from extremely pipe Bottom (can suitably increase centrifugation time), and complete soln, which is carefully poured into filter CS1, (please avoid pouring into a large amount of precipitatings and blocking Filter), push handle filtering is slowly pushed, filtrate is collected in the pipe of clean 50ml.
Note: should be mixed immediately after solution P4 is added, avoids generating localized precipitation.If pouring into filter CS1 after centrifugation In solution have white precipitate that will not influence to filter.If thallus is excessive (> 100m1), recommend to extend centrifugation time to 20- 30min
(7) isopropanol (isopropanol, which is added, is excessively easy to cause RNA to pollute) of 0.3 times of filtrate volume is added into filtrate, It turns upside down and is transferred in adsorption column CP6 (adsorption column is put into 50ml collecting pipe) after mixing.
Note: filtrate can lose after filtering, and the isopropanol of different volumes please be added according to the difference of loss.Adsorption column CP6 Maximum volume be 15ml, so needing a points of 2 times mistake columns.
(8) room temperature 8000rpm is centrifuged 2min, outwells the waste liquid in collecting pipe, adsorption column CP6 is placed back in collecting pipe In.
Note: crossing column for acquired solution in step 7 point 2 times, operated every time by conditions above.
(9) 10ml rinsing liquid PW (please first check whether and dehydrated alcohol has been added) is added into adsorption column CP6,8000rpm It is centrifuged 2min, the waste liquid in collecting pipe is discarded, adsorption column is placed back in collecting pipe.
(10) repetitive operation step 9.
(11) 3ml dehydrated alcohol is added into adsorption column CP6, room temperature 8000rpm is centrifuged 2min, outwells waste liquid.
(12) adsorption column CP6 is placed back in collecting pipe, 8000rpm is centrifuged 5min, it is therefore an objective to will be remaining in adsorption column Rinsing liquid removal.
Note: the residual of ethyl alcohol will affect subsequent enzymatic reaction (digestion, PCR etc.) experiment in rinsing liquid.Under ensuring Trip experiment is not influenced by residual ethanol, it is proposed that adsorption column CP6 uncaps, is placed in and is placed at room temperature for several minutes, thoroughly to dry suction Remaining rinsing liquid in enclosure material.
(13) adsorption column CP6 is placed in a clean 50ml collecting pipe, is vacantly added dropwise to the intermediate position of adsorbed film 1-2ml elution buffer TB, is placed at room temperature for 5min, then room temperature 8, and 000rpm (~8,228x g) is centrifuged 2min.By 50ml from Eluent in heart pipe all moves into a clean 1.5ml centrifuge tube, -20 DEG C of preservations.
2. carrying the preparation of P-miR-env recombinant plasmid CTS nanoparticle: being received using complex coacervation preparation CTS-P-miR-env P-miR-env (0.225ug/ μ L) of the 60 μ g without RNase is added in 1mL TPP solution, is uniformly mixed by the grain of rice；By P-miR- Env-TPP solution is instilled in 3mL chitosan-acetic acid solution with 2ml syringe needle point drop-wise, and rate of addition is after 40d-50d/min is instilled Whirlpool mixing 30s is incubated at room temperature 30min.Nanoparticle suspension is collected in no RNAse, the EP pipe without DNase.
3. nano-complex performance indexes detects:
(1) transmission electron microscope observing takes the CTS-P-miR-env nanoparticle one prepared to drop to the copper mesh for attaching carbon-fiber film On, 2 minutes are stood, blots suspension with filter paper, then 2% phosphotungstic acid counterstain 5min is added dropwise, is placed in air, in throwing after drying Penetrate observed under electron microscope.(2) particle size measurer analysis plus a small amount of CTS-P-miR-env nanoparticle solution are in analyzer sample introduction Kong Zhong, analysis measurement nanoparticle partial size.(3) measurement of nanoparticle encapsulation rate is control with blank CTS nanoparticle, and precision measures CTS-P-miR-env nanoparticle 0.5ml, UV spectrophotometer measuring OD260, this representation DNA total amount；Take appropriate CTS-P- MiR-env nanoparticle, under the conditions of 4 DEG C, the fixed centrifugation 20min of 13000 × g, precision measures supernatant 0.5ml, ultraviolet spectrometry light The light absorption value of 260 nanometers in degree meter detection supernatant, this represents free amount of DNA, then is calculated by the following formula encapsulation rate: Encapsulation rate G%=[(W total-W trip)/W is total] × 100%, W is always the total amount of DNA in formula；W trip is the DNA's to dissociate in supernatant Amount.Detect five samples.
4.DF-1 cell transfecting
It is divided into four groups by the difference of transfection reagent, according to illustrating for 12 orifice plate dosages:
A group is blank control group (cell does not apply any intervention)；
B group is naked p-miR-env recombinant plasmid control group；With 1.6 μ g of DNA content transfection；
C group is control liposome group (LipofeterTMLiposome) 1.6 μ gDNA+5 μ L transfection reagents；
D group is CTS-p-miR-env nanoparticle group, is respectively 1.6 μ g with DNA content, 3.2 μ g, 6.4 μ g transfection；Every time 2 repetitions are done in transfection.
Transfection: inhaling the cell culture fluid abandoned in 12 orifice plates before transfection, the DMEM culture solution of 1ml serum-free is added, and then will The CTS-p-miR-env nano-complex or gymnoplasm grain of different volumes are added in corresponding cell hole, shake culture plate, gently It mixes.
Culture: 12 well culture plates are placed in 5% CO2In incubator, after 37 DEG C of incubation 6h, inhales and abandon transfection liquid, be changed to DMEM culture solution containing 10%FBS, continues to cultivate.
(5) it is cultivated after transfecting
Growth plate is placed in the incubator of 5% CO2,37 DEG C of culture 24-96h, transfection is done every time repeats three times.
After transfection for 24 hours, the cell of 48h, 72h observation band green fluorescence under fluorescence inverted microscope, flow cytometer inspection Transfection efficiency is surveyed, MTT detects cell-proliferation activity, and Real-time PCR detects the expression of ALV-J mRNA in group of cells, Western blot detects viral envelope proteins expression.

Claims (1)

1. the ALV-J P-miRNA-env recombinant plasmid nano-complex based on chitosan package, it is characterised in that: the base In chitosan package ALV-J P-miRNA-env recombinant plasmid nano-complex be CTS-P-miRNA-env nano-complex, Preparation method is as follows:
CTS-P-miR-env nano-complex is prepared using complex coacervation: by the P- without RNase of 60 μ g 0.225ug/ μ L MiR-env is added in 1mg/ml TPP aqueous solution 1mL, is uniformly mixed；By P-miR-env-TPP solution 2ml syringe needle drop Shape instills in the chitosan-acetic acid solution that 3mL concentration is 2mg/ml, and rate of addition 40d-50d/min, whirlpool mixes after instillation 30s is incubated at room temperature 30 min；Collect nano-complex suspension in no RNAse, the EP pipe without DNase to obtain the final product；
Nano-complex particle size range obtained is at 90-110 nanometers.
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