CN109893664A - A kind of cation lipid nano particle/DNA compound and preparation method thereof - Google Patents

A kind of cation lipid nano particle/DNA compound and preparation method thereof Download PDF

Info

Publication number
CN109893664A
CN109893664A CN201810038987.4A CN201810038987A CN109893664A CN 109893664 A CN109893664 A CN 109893664A CN 201810038987 A CN201810038987 A CN 201810038987A CN 109893664 A CN109893664 A CN 109893664A
Authority
CN
China
Prior art keywords
nano particle
dna
cation lipid
compound
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810038987.4A
Other languages
Chinese (zh)
Other versions
CN109893664B (en
Inventor
彭飞
夏言富
靳开远
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan University
Original Assignee
CHENGDU JINKAI BIOLOGICAL TECHNOLOGY Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHENGDU JINKAI BIOLOGICAL TECHNOLOGY Co Ltd filed Critical CHENGDU JINKAI BIOLOGICAL TECHNOLOGY Co Ltd
Publication of CN109893664A publication Critical patent/CN109893664A/en
Application granted granted Critical
Publication of CN109893664B publication Critical patent/CN109893664B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention provides a kind of cation lipid nano particle/DNA compound and its preparation process, the preparation process the following steps are included: (1) by cationic lipid material by dissolving by heating in dehydrated alcohol；(2) ethanol solution that step (1) is prepared is added drop-wise in aqueous phase solution, is self-assembly of cation lipid nano particle；(3) remaining ethyl alcohol in step (2) cation lipid nano particle is removed；(4) it filters.(5) DNA solution is prepared；(6) DNA solution that the cation lipid nano particle and step (5) being prepared according to certain quality than mixing step (4) are prepared, to form cation lipid nano particle/DNA compound；(7) it filters.Preparation method of the present invention is easy to operate, quick, the cation lipid nano particle being prepared/DNA compound partial size is 50~150nm, PDI < 0.3, in monodispersity distribution, stable structure, and compound is in end-filtration degerming, effective guarantee safety of the compound in the clinical application that preparation treatment is tumour medicine.

Description

A kind of cation lipid nano particle/DNA compound and preparation method thereof
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of cation lipid nano particle/DNA compound and Preparation method.
Background technique
The successful key of gene therapy is can safely and effectively transmit therapeutic agent by carrier whether in vivo Into in target cell.Gene therapy vector is divided into viral vectors and non-virus carrier.Although viral vectors is passed as efficient Delivery system is with gene transfection and the therapeutic purposes of achieving the goal, but viral vectors contains the virus protein of immunogenicity, mesh because of it Gene useful load it is limited and at high price the problems such as so that the lipidic nanoparticles (lipid as non-virus carrier Nanoparticles, LNPs) due to its vitro stability is good, can be degraded in vivo, is safe and reliable the advantages that and closed extensively Note, and in the gene therapy research for the congenital and posteriority genetic defect being widely used in.LNPs refers to by a kind of or more The vesicles that kind lipid composition is formed, can be effectively compressed and transmit various nucleic acid molecules, from DNA, RNA to chromosome, even It is cell；LNPs is conducive to large-scale production because of the constructing plan that it is determined and the modification the features such as being easy to targeting ligand.
Cation lipid is a kind of amphipathic molecule, is mainly keyed hydrophobicity by chemistry by positively charged polar head Long-chain has the characteristics that be easily-synthesized and convenient for being modified in its each composition domain.Cation lipid nano particle can lead to nucleic acid Cross electrostatic adsorption and be formed by compound, can not only compress nucleic acid to increase its density, can also protect nucleic acid not by The degradation of nuclease, and by the way that electrostatic interaction occurs with negatively charged cell membrane to be effectively combined target cell, and then will Delivery of nucleic acids is into target cell and mediates high-caliber gene expression.
Cation lipid nano particle and DNA are formed by compound, i.e. cation lipid nano particle/DNA compound Its partial size is the principal element for influencing transfection efficiency of cells in vitro；The biggish cation lipid nano particle of partial size and partial size are smaller Compare, easily removed in vivo by blood circulation system, to influence to reach the DNA medication amount of target tissue or target cell.Shadow The principal element for ringing cation lipid nano particle/DNA compound partial size includes the composition of lipid, the ratio of lipid and nucleic acid, And preparation method thereof etc..
Currently, the preparation method of LNPs includes film dispersion method, reverse phase evaporation, alcohol injection, micro emulsion cooling method, freezes Melt method and freeze-drying etc..Wherein, alcohol injection be by the ethanol solution of lipid and/or drug at a suitable temperature, It is injected into the aqueous phase solution of high-speed stirred, ethyl alcohol is spread rapidly, is assembled due to thermodynamic (al) so as to cause lipid molecular Form LNPs.This method has the advantage that (1), instead of conventional organic solvents, chloroform, increases LNPs medication with ethyl alcohol Safety coefficient ensure that the quality of product；(2) oil containing lipid only need to be mutually directly injected into water phase and can be formed by this method LNPs, it is simple and convenient, it is easy to large-scale production；(3) this method mild condition is not easy to make degradation of lipid or oxidative deformation, reduce To LNPs structural damage；(4) LNPs partial size < 100nm made from alcohol injection is conducive to the phagocytosis and transfection of cell； (5) the potential drug toxicity of organic residual solvent can avoid by the ethyl alcohol of removal injection after LNPs is formed.Therefore, ethyl alcohol Injection method is increasingly becoming the large-scale method for producing being concerned.
However, there is also many restrictions sexual factors for common alcohol injection industrial at present, for example, the LNPs formed The ratio of granularity and homogeneity and the concentration of lipid soln, mixing speed, lipid composition and oil phase and water phase has much relations； In addition, alcohol injection is often by mechanical forces, by oily mutually according to certain volume than being injected into the water phase of respective volume with shape Continuous production can not achieve due to the limitation of water phase reservoir tank volume at LNPs, increase the difference between batch.Factors above Limit the large-scale production of LNPs.
LNPs prepared by alcohol injection or liposome often need to uniform step, and (such as high-pressure homogeneous, ultrasonic method is squeezed Method out) to improve the uniformity of nano particle, but still have certain office when using the extrusion molding for being most suitable for large-scale production It is sex-limited:
(1) for the liposome mini-extruder extrusion instrument of conventional 47mm in vertical extrusion process, filter membrane easily generates blocking, or even can shadow The quality to liposome or LNPs product by the gross is rung, liposome or the large-scale production of LNPs are seriously limited.
(2) in order to expand the treating capacity of sample, typically achieve the purpose that mass production by increasing membrane area, but It is to increase membrane area to need bigger extrusion device, not only production cost greatly improves, and the stability of extrusion cannot guarantee that.
It can be seen that still can be faced and be received using homogenization step when cation lipid nano particle is mass produced The problems such as rice grain particle diameter distribution is uneven and structural instability, and quality is difficult to control.
LNPs/ nucleic acid complexes are prepared frequently with the direct dissolution method of ethyl alcohol at present, i.e., ethyl alcohol are dissolved into cation lipid Directly be prepared cation lipid nano particle/nucleic acid complexes with the buffer mixing containing nucleic acid afterwards, in the method by Ethyl alcohol is removed before not mixing with nucleic acid, so certain influence can be generated to the structural stability of nucleic acid.Due to compound In with the presence of nucleic acid, therefore revolving method cannot be used to remove ethyl alcohol, and sample need to be removed using the mild mode such as ultrafiltration or dialysis In ethyl alcohol, but to handle the time long, complicated for operation for these modes, and sample loss is larger, and Residual ethanol also compares in sample Revolving method it is higher.And the influence for needing to use dilution method immediately after mixing to reduce ethyl alcohol to complex stabilities, therefore it is unfavorable It is controlled in the quality of product.If not removing the ethyl alcohol in sample, and it will affect safety of the compound in terms of clinical application.
Using cation lipid nano particle prepared by alcohol injection, even if still can not using homogenization step Solve cationic nano-grain there is a situation where it is heterogeneous, due to contain a fairly large number of large-size particles, not only make polydispersion Property increase, also make the partial size of obtained cation lipid/nucleic acid complexes after mixing with nucleic acid larger, 0.22 can not be passed through μm filter membrane makes product be filtered degerming in terminal, therefore increases the risk of clinical application.Due to not being available terminal The method of filtration sterilization, so need aseptically to operate during preparing cation lipid/nucleic acid complexes, by This increases production cost, and quality controllability is poor.
During preparing cation lipid nano particle/nucleic acid complexes, often pass through the active force pumped for nucleic acid to drip The mode added is injected into the cation lipid nano particle of alcohol injection preparation, and is kept stirring to form cation lipid Nano particle/nucleic acid complexes.The method has the following problems: firstly, cation lipid nano particle and nucleic acid are mixing In the process due to stirring, there are the effects of shearing force cannot reach effective so that the two immixture time is short It is compound, to form stable compound；Secondly, compound local concentration is higher in order to prevent and partial size is big and homogeneity is poor, because The speed that nucleic acid is added dropwise in this is slower, and the production time is long；Third, the method are limited to the size and its stirring power of blender volume Power, and the normal > 220nm of cation lipid nano particle/nucleic acid complexes average grain diameter being prepared, can not accomplish compound Object end-filtration degerming, therefore need to carry out in superclean bench or isolator using this method, this method is not applicable as a result, In large-scale production and preparation cation lipid nano particle/nucleic acid complexes.
Therefore, it is compound how to prepare the uniform cation lipid nano particle/nucleic acid with monodispersity of particle size Object and preparation process can experimental size metaplasia produce and clinical demand and the safety for ensureing clinical application, be mesh The problem of technical study urgent need to resolve of preceding cation lipid nano particle/nucleic acid complexes.
Summary of the invention:
It is an object of the invention to provide a kind of ethanol injection legal system of improvement from the deficiencies in the prior art are solved The method of standby cation lipid nano particle/DNA compound, it is specific: to be not less than using improvement alcohol injection preparation concentration After 6mg/ml cation lipid nano particle, ethyl alcohol is removed by revolving method, without homogenization (homogenization) step Suddenly, after mixing with DNA solution, cation lipid nano particle/DNA compound is can be obtained in filtration sterilization.Preparation method behaviour Make simply, fast, can not only guarantee cation lipid nano particle diameter < 100nm, and particle size is uniform, and has Monodispersity, and obtained cation lipid nano particle/DNA compound particle diameter distribution is uniform, stable structure, and ethyl alcohol is residual Allowance meets medicinal standard and Product Terminal filtration sterilization, and the effective guarantee compound is tumour medicine in preparation treatment Clinical application in safety.
The purpose of the invention is achieved by the following technical solution:
Present invention firstly provides a kind of preparation methods of cation lipid nano particle/DNA compound, including cation Preparation method and cation lipid nano particle/DNA compound preparation method of lipidic nanoparticles.
Wherein, the preparation method of the cation lipid nano particle, comprising the following steps:
(1) alcohol injection: cationic lipid material is dissolved in dehydrated alcohol, is dissolved by heating；
(2) ethanol solution containing cationic lipid material that above-mentioned steps (1) are prepared is added drop-wise to aqueous phase solution In, it is self-assembly of cation lipid nano particle；
(3) remaining ethyl alcohol in above-mentioned steps (2) cation lipid nano particle is removed；
(4) it filters；
Cation lipid nano particle/DNA compound preparation method, comprising the following steps:
(5) DNA solution is prepared；
(6) the cation lipid nano particle that is prepared according to certain mass ratio mixing above-mentioned steps (4) and above-mentioned The DNA solution that step (5) is prepared, to form cation lipid nano particle/DNA compound；
(7) it filters；
Concentration of the cationic lipid material in dehydrated alcohol described in step described in above-mentioned preparation method (1) is 30 ~100mg/ml, preferably 50-60mg/ml；The volume ratio of ethanol solution liquid compatible with water described in step (2) is 1:3~1: 6, final concentration of 6~25mg/ml of the cationic lipid material in ethyl alcohol and water phase mixed solution；Form cation Lipidic nanoparticles/DNA compound partial size is 50~150nm, compound dispersion index PDI < 0.3.
When being greater than 100mg/ml due to concentration of the cation lipid in dehydrated alcohol, then particle size < can not be formed The cation lipid nano particle of 150nm, PDI < 0.3；However, concentration of the cationic lipid material in dehydrated alcohol is less than When 30mg/ml, since the concentration value of cationic lipid material is small, it is also easy to produce heterogeneous cation lipid nano particle, and more Dispersibility increases.
6~25mg/ml cation lipid the nano particle being prepared in above-mentioned preparation method using alcohol injection, nothing The step of homogenization need to be further carried out, can be obtained in monodispersity, uniform, partial size is 50~150nm cationic lipid Matter nano particle.
The 0.3 cation lipid nano particle of partial size < 150nm and PDI < that above-mentioned preparation method is prepared is suitable for making For the transport vehicle of DNA, it not only can be further improved the compression of cationic lipid material and stabilized DNA transfection be conveyed into the cell Effect, and be convenient for 0.22 μm of membrane filtration, it is quality controllable.
Wherein, cation lipid described in above-mentioned preparation method is selected from (2,3-Dipropyloxypropyl)trimethylammonium chloride (DOTAP), N- [1- (2,3- dioleoyl chlorine) propyl]-N, N, N- trimethyl ammonium chloride (DOTMA), trifluoroacetic acid dimethyl -2,3- Two oily alkenyloxy group propyl -2- (2- spermine formamido group) ethyl ammoniums (DOSPA), bromination trimethyldodecane base ammonium (DTAB), bromine Change trimethyl tetradecyl base ammonium (TTAB), trimethylcetyl base ammonium (CTAB) or bromoethyl dibasecylammonium bromide At least one of (DDAB), preferred cation lipid is DOTAP.
The DOTAP is a kind of common cation lipid, since with DNA or RNA to form stable transfection compound for it Object enters cell, and nucleic acid is released into the cell, therefore, can be used as a kind of eukaryotic cell transfection reagent for eukaryocyte The carrier of transfection and DNA vaccination.
Cation lipid nano particle described in step described in above-mentioned preparation method (6) and the DNA mass ratio For 6:1~125:1, preferably 6:1~20:1.
When the cation lipid nano particle and the DNA are with mass ratio 6:1~125:1, formed cationic lipid Matter nano particle/DNA compound partial size is 50~150nm, PDI < 0.3；In particular, working as the cation lipid nano particle When with the DNA with mass ratio 6:1~20:1, formed cation lipid nano particle/DNA compound partial size < 100nm, PDI < 0.3, structure more stable homogeneous more conducively play the effect for inhibiting growth of tumour cell.When the two mass ratio is less than 6:1 When, DNA cannot be completely compound with cation lipid nano particle.Due to the cation lipid nano particle of the method for the present invention preparation Maximum concentration is about 25mg/ml and cation lipid nano particle/DNA compound its DNA concentration in terms of druggability is general Greater than 0.1mg/ml, therefore, cation lipid nano particle and the isometric compound preceding DNA concentration of DNA are greater than 0.2mg/ml.By This, the mass ratio of cation lipid nano particle and DNA are up to 125:1.
Wherein, cationic lipid material described in above-mentioned preparation method is the mixing of the cation lipid and helper lipids Object, helper lipids are selected from phosphatidyl-ethanolamine (PE), phosphatidyl choline (PC), cholesterol (Chol) or dioleoyl phosphatidyl second At least one of hydramine (DOPE).
Further, cation lipid in the mixture of cation lipid described in above-mentioned preparation method and helper lipids It is greater than 1:1 with the mass ratio of helper lipids.
The stability of cation lipid nano particle can be improved in helper lipids.Common helper lipids, as cholesterol exists In dehydrated alcohol, in dehydrated alcohol, solubility at 50 DEG C is about the solubility about 33.6mg/ml or DOPE at 50 DEG C 36.5mg/ml, so when adding helper lipids in cation lipid, when the mass ratio > 1:1 of the two, can be obtained partial size be 40~ 100nm is in monodispersity (PDI < 0.3) cation lipid nano particle；On the other hand, when in cation lipid nano particle not When containing helper lipids, cation lipid nano particle and DNA can equally be maintained to be formed by the stability of compound.So And as mass ratio≤1:1 of cation lipid and helper lipids, prepared cation lipid nano particle can not expire simultaneously Sufficient partial size is less than 150nm and PDI < 0.3.
Cation lipid nano particle/DNA compound described in step described in above-mentioned preparation method (6) can choose It is prepared in the semiautomatic plant containing t connector.The semiautomatic plant of the t connector is selected from such as Fig. 1, figure It is combined the unit shown in 2a or Fig. 2 b, wherein device shown in FIG. 1 is according to arrow direction by constant flow pump, sterile filters, liquid storage Bottle, constant flow pump, t connector are connected in sequence；Device shown in Fig. 2 a is successively connected by constant flow pump, T-type in the direction of arrows Device and liquid storage bottle containing air filter and its connecting pipe composition；Device shown in Fig. 2 b is from right to left successively by injecting Pump, syringe, t connector and liquid storage bottle composition；Preferred device is device shown in FIG. 1.
Cation lipid nano particle/DNA compound preparation method in the step (6) includes according to Fig. 1, Fig. 2 a Or the semiautomatic plant containing t connector is first assembled shown in Fig. 2 b, the cation lipid that then step (4) is prepared The DNA solution that nano particle and step (5) are prepared is respectively placed in the container of described device with the mass ratio of 6:1~125:1 It is interior, it is added in new container in t connector with stream after the mixing of 20~100ml/min speed and forms the cation lipid Nano particle/DNA compound.
The mixing velocity can obtain partial size < 150nm and uniform (the PDI < of size in 20~100ml/min 0.3) cation lipid nano particle/DNA compound, and mixing velocity is higher within this range, the partial size of compound is smaller, Homogeneity is higher；As mixing velocity < 20ml/min, cation lipid nano particle and DNA solution are when t connector mixes Easily assemble, to influence the particle size and uniformity of the compound, is unfavorable for the quality control of compound；Work as mixing When speed > 100ml/min, be limited to constant flow pump and connecting pipe in device, cannot achieve cation lipid nano particle and DNA solution mixes in t connector.
The semiautomatic plant containing t connector, on the one hand, structure is simple, is easily assembled；It on the other hand, can be with It can repeat steady production by easy steps and prepare the cation lipid of partial size < 150nm and size uniform (PDI < 0.3) to receive Rice grain/DNA compound, and do not have to the successive suitable of the dropwise addition for considering the cation lipid nano particle and the DNA solution Sequence.Further, the device as shown in Figure 1 can carry out whole before cation lipid nano particle and DNA solution are compound Body high-temperature sterilization, even if under the production environment of the non-cleaning such as the valuable instrument and equipments such as no isolator and super-clean bench, it can also be with It is compound to guarantee that cation lipid nano particle and DNA solution carry out in a simple closed sterile device context, not only The cation lipid nano particle having saved production cost, easily having accomplished scale production, and be prepared/DNA compound grain Diameter < 150nm and particle size is uniform (PDI < 0.3).
Step described in above-mentioned preparation method (1) heats preferred heating water bath, and the temperature of heating is 40~60 DEG C, and preferably 50 ~60 DEG C；When bath temperature is room temperature, the distribution of cation lipid nano particle is mixed and disorderly, but with the raising of bath temperature, sun The particle size of cationic lipid nano particle and distribution tend towards stability, and especially reach best at 50~60 DEG C.
Aqueous phase solution described in step described in above-mentioned preparation method (2) is aqueous solution, or the aqueous solution containing carbohydrate, The carbohydrate is selected from lactose, maltose, sucrose, glucose or trehalose, and the concentration of carbohydrate aqueous solution is 2-20%, preferably 4- 10%.
The method of removal ethyl alcohol is selected from revolving, dialysis, ultrafiltration, spray drying in step described in above-mentioned preparation method (3) Or freeze-drying etc., preferably rotate method.
Residual ethanol meets medicinal standard 0.5% in cation nanometer lipid granule after revolving.
It is filtered in step described in above-mentioned preparation method (4) and step (7) using 0.22 μm of membrane filtration, filtering Play the role of degerming simultaneously.
The length of the DNA is 100~2500bp.
DNA solution described in step described in above-mentioned preparation method (5) is aqueous dna.
Cation lipid nano particle described in step described in above-mentioned preparation method (6) is above-mentioned steps (4) preparation Obtained cation lipid nano particle is obtained after water dilutes, and the concentration after dilution is 1~4mg/ml.
Cation lipid nano particle prepared by the method for the present invention is suitble to and DNA formation particle size is uniform, is in single point Dissipate the compound of property.Before compound, it is 1~4mg/ml that cation lipid nano particle, which is diluted to concentration, not only by simple Preparation method can be realized, and can need to add different auxiliary materials according to different preparations, be conducive to carry out subsequent preparation process And large-scale production.
DNA described in step described in above-mentioned preparation method (5) is that plasmid or its variant or load have other DNA Plasmid；Either mitochondrial DNA or mitochondria DNA fragment.
Further, DNA described in step described in above-mentioned preparation method (5) is plasmid, selected from pVAX1, At least one of pcDNA3.1, pBR322 or pUC18；
DNA described in step described in above-mentioned preparation method (5) can also be the variant of pVAX1, i.e., reconstructed rear institute Formed the reproducible plasmid with most basic structural unit, include kalamycin resistance gene, pUC origin sequence and Plasmid backbone sequence, the nucleotide sequence of pMVA plasmid is as shown in SEQ ID NO:1；
DNA described in step described in above-mentioned preparation method (5) can also be in the specific nucleosides of pMVA plasmid Sour site carries out base mutation or missing, and to obtain pMVA-1 plasmid, nucleotide sequence is as shown in SEQ ID NO:2.
DNA described in step described in above-mentioned preparation method (5) is the plasmid that load has other DNA, is selected from nucleotide Sequence is as shown in NO.6~11 SEQ ID or nucleotide sequence and SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, sequence shown in SEQ ID NO.10 or SEQ ID NO.11 have in 90% or more the plasmid of homology At least one,
It is preferred that DNA is mitochondrial DNA or mitochondria DNA fragment.The nucleotide sequence of the mitochondria DNA fragment is such as Shown in NO:3~5 SEQ ID or its nucleotide sequence and sequence shown in NO.3~5 SEQ ID have 90% or more it is homologous Property.Another object of the present invention is to provide a kind of cation lipid nano particle/DNA compound, the compound passes through Aforementioned preparation process is prepared, and formed cation lipid nano particle/DNA compound partial size is 50~150nm, compound Dispersion index PDI < 0.3.
Wherein, the cation lipid nano particle/DNA compound is preferably DOTAP nano particle/pMVA-1 compound Object.
Cation lipid nano particle/DNA composite structure that above-mentioned preparation method is prepared is uniform, stablizes, and is applicable in It is filtered degerming in the terminal prepared in technique, it is controllable to not only contribute to product quality, and compound can be improved to swollen The inhibitory activity of tumor cell growth.
The present invention have the following advantages compared with the existing technology and the utility model has the advantages that
1, a kind of preparation method of cation lipid nano particle/DNA compound, the sun that this method is prepared are provided Cationic lipid nano particle diameter < 150nm, and distribution is uniform, has monodispersity, and inhibits activity of tumor cells height etc. Advantage.
2, a kind of preparation method of cation lipid nano particle/DNA compound, the sun that this method is prepared are provided Cationic lipid nano particle/DNA compound partial size < 150nm, and composite structure is stable and quality controllable.
3, a kind of preparation method of cation lipid nano particle/DNA compound, the sun that this method is prepared are provided Cationic lipid nano particle/DNA compound Determination of Residual Organic Solvents meets medicinal standard, and the compound can be ensured as tumour medicine Safety of the object in clinical application.
4, a kind of preparation method of cation lipid nano particle/DNA compound is provided, this method can contain in one kind Have and carry out preparing cation lipid nano particle/DNA compound in the semiautomatic plant of t connector, described device has knot The simply easy-to-assemble characteristic of structure, and it is capable of providing a simple closed sterile device context, it can be weighed by easy steps Multiple steady production prepares cation lipid nano particle/DNA compound of partial size < 150nm and size uniform (PDI < 0.3), And the sequencing for considering the dropwise addition of the cation lipid nano particle and the DNA solution is not had to.Make in said device Use this method that can realize the large-scale production of the compound as anti-tumor drug, to meet the needs of clinical application.
5, provide it is a kind of cation lipid nano particle is prepared using alcohol injection, without uniforming step, side Method is simple to operation, has saved production cost；Ethyl alcohol is eliminated with regard to revolving during preparing cation lipid nano particle, Ethyl alcohol is removed compared with after the completion of cation lipid nano particle/DNA compound preparation, is more advantageous to and saves production time and guarantor The quality control for hindering the compound, is prepared as tumour medicine conducive to the compound and is applied in clinic.
6, a kind of preparation method of cation lipid nano particle/DNA compound is provided, this method makes described compound Produce product can be realized end-filtration degerming, so that the quality of the compound clinical application obtains strong guarantee.
7, a kind of preparation method of cation lipid nano particle/DNA compound is provided, what this method was prepared answers Object partial size < 150nm is closed, the gene therapy of systemic applications is suitble to.
Detailed description of the invention:
Fig. 1, using preparation method of the present invention, prepare cation lipid nano particle/DNA compound using device 1 and show It is intended to, the device is according to arrow direction successively by constant flow pump, sterile filters, the liquid storage bottle 1 and 2 containing air filter, constant current Pump, t connector and liquid storage bottle 3 form；Wherein, the component expression of wire mark part can prepare cation lipid nanometer Assemble simultaneously high pressure sterilizing before particle/DNA compound, to maintain gnotobasis；
Fig. 2, using preparation method of the present invention, prepare cation lipid nanometer using device 2 (Fig. 2 a) and device 3 (Fig. 2 b) Particle/DNA compound schematic diagram, wherein device 2 shown in Fig. 2 a in the direction of arrows successively by constant flow pump, t connector and Liquid storage bottle containing air filter and its connecting pipe composition；Device shown in Fig. 2 b is from right to left successively by syringe pump, note Emitter, t connector and liquid storage bottle composition；
DOTAP nano particle/DNA compound agarose gel electrophoresis detection figure of Fig. 3, different quality ratio；
The obtained DOTAP nano particle/pMVA-1 compound of Fig. 4, preparation method of the present invention (DOTAP nano particle with PMVA-1 mass ratio be 10:1) particle size and its distribution；
The preparation-obtained cation lipid nano particle/DNA compound of Fig. 5, preparation method of the present invention is infused in ethyl alcohol Enter method uses the nano particle of cation lipid prepared by high-pressure homogenization step/DNA compound to inhibit A549 cell activity later Detection figure, wherein ■ indicates preparation method (referred to as new process) of the present invention, ● indicate after alcohol injection using high pressure Homogenizing step (referred to as old technology)；
Fig. 6, cation lipid nano particle/DNA compound inhibit the tumour growth of cervical carcinoma subcutaneous tumors model mice, In, ▼ indicates preparation method (referred to as new process) of the present invention, and ■ indicates (to claim after alcohol injection using high-pressure homogenization step For old technology)；
Fig. 7, use cation lipid nano particle/DNA prepared by high-pressure homogenization step multiple after alcohol injection Close the particle size and its distribution of object；
The preparation-obtained cation lipid nano particle/DNA compound of Fig. 8, preparation method of the present invention and ethyl alcohol are direct The cation lipid nano particle that dissolution method is prepared/DNA compound inhibits the detection figure of A549 cell activity, wherein ▲ Indicate preparation method (referred to as new process) of the present invention, ▼ indicates the direct dissolution method of ethyl alcohol.
The DOTAP nano particle and DNA shape of Fig. 9, preparation method of the present invention preparation different final concentrations in ethanol water At compound inhibit A549 cell activity detection figure, wherein ● indicate DOTAP in dehydrated alcohol concentration be 50mg/ml, Ethyl alcohol and water volume ratio are 1:5 (final concentration of the DOTAP in ethanol water is about 8.3mg/ml) preparation-obtained DOTAP The compound (sample) that nano particle and DNA are formed；■ indicates that DOTAP concentration in dehydrated alcohol is 20mg/ml, ethyl alcohol and water Volume ratio be 1:4 (final concentration of the DOTAP in ethanol water is about 4mg/ml) preparation-obtained DOTAP nano particle with The compound (reference substance 1) that DNA is formed；▲ indicate that DOTAP concentration in dehydrated alcohol is 100mg/ml, ethyl alcohol and water volume ratio For the preparation-obtained DOTAP nano particle of 1:2 (final concentration of the DOTAP in ethanol water is about 33.3mg/ml) and DNA The compound (reference substance 2) of formation.
Specific embodiment
Technical solution of the present invention is further detailed below by way of specific embodiment.It should be noted that Some common molecular biology manipulations and the common operation for preparing pharmaceutical preparation involved in the present invention, art technology Personnel can combine the existing textbook in this field, handbook and relevant device and examination on the basis of reading description of the invention The operation instruction of agent is completed.
Below with reference to embodiment and attached drawing, the present invention is described in further detail, but the embodiment invented is not limited to This.
The preparation and its characterization of embodiment 1, DOTAP nano particle
1, the preparation of DOTAP nano particle
(1) as shown in table 1, the DOTAP for weighing different quality is put into 250ml PETG (polyethylene terephthalate Ester -1,4-CHDM ester) in bottle, it is added in ethanol solution, the heating water bath in 50 DEG C, it gently shakes to accelerate DOTAP is completely dissolved.
(2) the good peristaltic pump of frame, setting speed are 6~8rpm, and the ethyl alcohol containing DOTAP that step (1) is prepared is molten Liquid is added drop-wise in aqueous phase solution with the rate of 30~120ml/min, and the total volume of ethanol solution liquid compatible with water is 60ml, ethyl alcohol The volume ratio of solution liquid compatible with water is as shown in table 1.Drop outlets are maintained at 5cm or so away from liquid level, and DOTAP is in ethanol water In can be self-assembly of DOTAP nano particle.
(3) it is added drop-wise to DOTAP ethanol solution in step (2) after being completed in aqueous phase solution, dehydrated alcohol flushing pipe With PETG bottles.After the completion of flushing, continue to be dispersed with stirring system 10min with 140rpm.
(4) vacuum distillation removal ethyl alcohol:
Rotary evaporator is opened, sets bath temperature as 40 DEG C；Coolant circulation pump is opened, sets cryogenic temperature as 5 DEG C.It will The DOTAP nano particle dispersion solution of step (3) preparation is transferred in revolving bottle, is evaporated under reduced pressure；Vacuum pump is opened, rotation is closed and steams Send out all rotation mouths of device.After the waste liquid collected in bottle to be distilled is greater than 120ml, stop distillation.Liquid in revolving bottle is transferred to newly PETG bottle in, be settled to 500ml with pure water.
(5) the DOTAP nano particle after step (4) to be rotated to removal ethyl alcohol is saved by 0.22 μm of membrane filtration, spare.
2, the characterization of DOTAP nano particle:
(1) test sample of DOTAP nano particle is prepared:
Sterile purified water is added in the DOTAP nano particle of the various concentration of above-mentioned steps preparation, high speed vibrates, and makes It is sufficiently dissolved, and is stood at room temperature.
(2) partial size of DOTAP nano particle is detected
Nanometer particle size potentiometric analyzer (Malvern is added in the DOTAP nano particle test sample that step (1) is prepared Zetasizer Nano ZS) planchet in, be put into test trough, setting equilibration time is 1min, each sample parallel testing 3 Group data.Obtain the average grain diameter of composite sample.
Testing result is as shown in table 1, and the data fit partial size for underlining part is less than 150nm, thus PDI < 0.3 may be used See, the solubility of DOTAP in ethanol is 30~100mg/ml (especially 50~60mg/ml), the body of ethyl alcohol liquid compatible with water Product is than being 1:3~1:6, then final concentration of 6~25mg/ml of the DOTAP in ethyl alcohol and water phase mixed solution, is prepared Partial size 50~150nm, the PDI < 0.3 of DOTAP nano particle.
Nanometer formed in various concentration and different ethanol water volume ratios of the table 1.DOTAP in dehydrated alcohol The partial size and PDI value of particle
"-" indicates that obviously not meeting cation lipid nano particle/DNA compound partial size is 50~150nm, PDI < 0.3 Requirement.
The preparation and its characterization of embodiment 2, DOTAP/ cholesterol (DOTAP/Chol) nano particle
1, the preparation of DOTAP/Chol nano particle:
(1) 33.6mg DOTAP and the 33.6mg cholesterol (mass ratio 1:1) weighed respectively is respectively put into 250ml It in PETG bottles, is added in ethanol solution, the heating water bath in 50 DEG C, gently shakes to accelerate the complete of DOTAP and cholesterol Fully dissolved.
(2) the good peristaltic pump of frame, setting speed are 6~8rpm, and the ethyl alcohol containing DOTAP that step (1) is prepared is molten Liquid is added drop-wise in aqueous phase solution with the rate of 30~120ml/min, wherein the volume ratio of ethanol solution liquid compatible with water is 1:3 Or 1:4, drop outlets are maintained at 5cm or so away from liquid level, DOTAP and cholesterol can be self-assembly of in ethanol water DOTAP/Chol nano particle.
(3) with 1 step of embodiment (3).
(4) with 1 step of embodiment (4).
2, the characterization of DOTAP/Chol nano particle:
The method that method is characterized with DOTAP nano particle in embodiment 1.
Testing result is as shown in table 2, and as DOTAP and cholesterol mass ratio > 1:1, ethyl alcohol liquid volume ratio compatible with water is 1: When 3~1:6, partial size < 150nm, the PDI < 0.3 of obtained DOTAP/Chol nano particle.
The preparation and representation of embodiment 3, DOTAP/DOPE nano particle
1, the preparation of DOTAP/DOPE nano particle:
(1) the 36.5mg DOTAP and 36.5mgDOPE (mass ratio 1:1) that weigh respectively are respectively put into 250ml It in PETG bottles, is added in ethanol solution, the heating water bath in 50 DEG C, gently shakes that DOTAP's and DOPE is complete to accelerate Dissolution.
(2) the good peristaltic pump of frame, setting speed are 6~8rpm, and the ethyl alcohol containing DOTAP that step (1) is prepared is molten Liquid is added drop-wise in aqueous phase solution with the rate of 30~120ml/min, wherein the volume ratio of ethanol solution liquid compatible with water is 1:3 Or 1:4, drop outlets are maintained at 5cm or so, DOTAP and DOPE away from liquid level can be self-assembly of DOTAP/ in ethanol water DOPE nano particle.
(3) with 1 step of embodiment (3).
(4) with 1 step of embodiment (4).
2, the characterization of DOTAP/DOPE nano particle:
The method that method is characterized with DOTAP nano particle in embodiment 1.
Testing result is as shown in table 2, and as DOTAP and DOPE mass ratio > 1:1, ethyl alcohol liquid volume ratio compatible with water is 1:3 When~1:6, partial size < 150nm, the PDI < 0.3 of obtained DOTAP/DOPE nano particle.
Partial size and PDI value of the table 2DOTAP/ helper lipids nano particle in different ethanol water volume ratios
The stability of cation lipid nano particle can be improved in helper lipids.Common helper lipids, as cholesterol exists In dehydrated alcohol, in dehydrated alcohol, solubility at 50 DEG C is about the solubility about 33.6mg/ml or DOPE at 50 DEG C 36.5mg/ml, so when adding helper lipids in cation lipid, when the mass ratio > 1:1 of the two, can be obtained partial size be 40~ 100nm is in monodispersity (PDI < 0.3) cation lipid nano particle.
And as mass ratio≤1:1 of cation lipid and helper lipids, prepared cation lipid nano particle without Method meets partial size less than 150nm and PDI < 0.3 simultaneously.
Embodiment 4, using device 1, with different mixing velocities prepare cation lipid nano particle/DNA compound and It is characterized
1, using device 1, cation lipid nano particle/DNA compound is prepared with different mixing velocities
(1) solution for preparing pMVA-1 (SEQ ID NO:2) and other plasmids, as pMVA-2 (SEQ ID NO:6), PMVA-3 (SEQ ID NO:7), pMVA-4 (SEQ ID NO:8) and pMVA-5 (SEQ ID NO:9), pMVA-6 (SEQ ID ) and pMVA-7 (SEQ ID NO:11) NO:10.
(2) by liquid storage bottle 1,2,3, sterile filters and its between pipeline according to being connected shown in Fig. 1 after, carry out integral high-temperature and go out Bacterium, and be sequentially connected to form a closed gnotobasis with constant flow pump.
(3) as shown in Figure 1, prepared by the embodiment 1-3 cation lipid nano particle being prepared and above-mentioned steps (1) Obtained DNA solution is respectively placed in beaker with mass ratio 10:1, is sterile filtered by constant flow pump and sterile filters to liquid storage In bottle 1 and liquid storage bottle 2, after aseptic filtration to be done, then respectively by constant flow pump, according to shown in table 3, with 5~100ml/min's Speed mixes in t connector, and is added dropwise in liquid storage bottle 3, stands 30min, can be compounded to form the cationic lipid Matter nano particle/DNA compound.
(4) 0.22 μm of membrane filtration degermings.
2, the cation lipid nano particle/DNA compound characterization being prepared with different mixing velocities
(1) cation lipid nano particle/DNA compound test sample is prepared:
Sterile purified water is added to the cation lipid nanometer that above-mentioned steps 1 are prepared with different mixing velocities In grain/DNA compound, high speed vibrates, and dissolves compound, stands at room temperature.
(2) cation lipid nano particle/DNA compound partial size is detected
Nanoparticle is added in the cation lipid nano particle that above-mentioned steps (1) are prepared/DNA compound test sample In the planchet of diameter potentiometric analyzer (Malvern Zetasizer Nano ZS), it is put into test trough, setting equilibration time is 1min, 3 groups of data of each sample parallel testing.
Testing result is as shown in table 3, when mixing velocity is in 20~100ml/min, can obtain partial size < 150nm and big Cation lipid nano particle/DNA the compound of small uniform (PDI < 0.3), and mixing velocity is higher within this range, it is compound The partial size of object is smaller, and homogeneity is higher；As mixing velocity < 20ml/min, cation lipid nano particle and DNA solution are in T-type Connector is easily assembled when mixing, compound partial size > 150nm and homogeneity is poor (PDI > 0.3)；As mixing velocity > When 100ml/min, it is limited to constant flow pump and connecting pipe in device, cannot achieve cation lipid nano particle and DNA solution It is mixed in t connector.
Equally, use device 2 or device 3, and cation lipid nano particle and DNA solution are set in t connector Mixing velocity be 20~100ml/min when, also can guarantee cation lipid nano particle/DNA compound partial size < 150nm, PDI < 0.3.
Table 3. utilizes device 1, prepares cation lipid nano particle/DNA compound partial size with different mixing velocities And PDI value
Embodiment 5, the cation lipid nano particle/DNA compound and its table that different quality ratio is prepared using device 1 Sign:
1, cation lipid nano particle/DNA compound is prepared using device 1
(1) solution for preparing pMVA-1 (SEQ ID NO:2) and other plasmids, as pMVA-2 (SEQ ID NO:6), PMVA-3 (SEQ ID NO:7), pMVA-4 (SEQ ID NO:8) and pMVA-5 (SEQ ID NO:9), pMVA-6 (SEQ ID ) and pMVA-7 (SEQ ID NO:11) NO:10.
(2) by liquid storage bottle 1,2,3, sterile filters and its between pipeline according to being connected shown in Fig. 1 after, carry out integral high-temperature and go out Bacterium, and be sequentially connected to form a closed gnotobasis with constant flow pump.
(3) DNA that cation lipid nano particle and above-mentioned steps (1) that embodiment 1-3 is prepared are prepared Solution is respectively placed in beaker as shown in Figure 1 with mass ratio 1:1~125.1:1, passes through constant flow pump and the sterile mistake of sterile filters Filter is into liquid storage bottle 1 and liquid storage bottle 2, after aseptic filtration to be done, then respectively by constant flow pump, with the speed of 50ml/min in T It mixes, and is added dropwise in liquid storage bottle 3 in type connector, stand 30min, the cation lipid nanometer can be compounded to form Grain/DNA compound.
(4) 0.22 μm of membrane filtration degermings.
2, cation lipid nano particle/DNA compound agarose gel electrophoresis detection of different quality ratio
(1) it prepares 1% Ago-Gel: weighing agarose and be placed in conical flask, 1 × TAE is added, microwave stove heating is boiled All melt to agarose, DNA dyestuff Golden View is added, shakes up, is prepared as 1.0% Ago-Gel liquid.
(2) it prepares gel slab: after preparing gel slab, the Ago-Gel prepared in step (1) being cooled to 65 DEG C, Enter on inside groove glass plate, is formed and glue-line.At room temperature stand until gel solidify completely, gel and inside groove are put into electrophoresis tank In.1 × TAE electrophoretic buffer is added until not crossing offset plate 1-2mm.
(3) it is loaded: being respectively 1:1,6:1,10:1,15:1 and 20:1 by cation lipid nano particle and DNA mass ratio Cation lipid nano particle/DNA compound test sample, be added to after being mixed respectively with sample-loading buffer in step (2) In the gel pore prepared.
(4) electrophoresis: the gel slab after sample-adding is powered immediately carries out electrophoresis.When bromophenol blue is moved to apart from offset plate lower edge about When at 1cm, stop electrophoresis.
(5) preservation of taking pictures is carried out using gel imaging system.
Cation lipid nano particle/DNA compound agarose gel electrophoresis of different quality ratio detects, such as Fig. 3 institute Show, show in cation lipid nano particle/DNA compound object the mass ratio of DOTAP nano particle and DNA be respectively 6:1, When 10:1,15:1 and 20:1, it can effectively be detained DNA under agarose gel electrophoresis effect.And there is DNA and dissociates in ratio 1:1 The case where, it is difficult to form satisfactory cation lipid nano particle/DNA compound.
3, cation lipid nano particle/DNA compound characterization of different quality ratio:
Method is the same as the cation lipid nano particle/DNA compound being prepared in embodiment 4 with different mixing velocities Characterizing method.
Testing result as shown in table 4 and figure 4, obtain composite sample average grain diameter be 50~150nm, PDI < 0.3, And the compound ratio about 60.12% of partial size >=220nm compound ratio < 0.01%, partial size < 150nm.
Cation lipid nano particle/DNA compound the partial size and PDI value of 4. different quality ratio of table
Embodiment 6 prepares cation lipid nano particle/DNA compound and its characterization using device 2:
1, cation lipid nano particle/DNA compound is prepared using device 2
(1) solution for preparing pMVA-1 (SEQ ID NO:2) and other plasmids, as pMVA-2 (SEQ ID NO:6), PMVA-3 (SEQ ID NO:7), pMVA-4 (SEQ ID NO:8) and pMVA-5 (SEQ ID NO:9), pMVA-6 (SEQ ID ) and pMVA-7 (SEQ ID NO:11) NO:10.
(2) according to shown in Fig. 2 a, it is 1 that cation lipid nano particle prepared by embodiment 1-3, which is diluted with water to concentration, After~4mg/ml, mark " cation lipid is respectively placed in mass ratio 1:1~125:1 with the plasmid of above-mentioned steps (1) preparation In nano particle " and the liquid storage bottle of " DNA ", by constant flow pump by the cation lipid nano particle and DNA in two liquid storage bottles Solution is pumped simultaneously with the speed of 50ml/min and is mixed into t connector, and is added dropwise in liquid storage bottle, is stood 30min, is obtained To cation lipid nano particle/DNA compound.
(3) 0.22 μm of membrane filtration degermings.
2, cation lipid nano particle/DNA compound characterization that use device 2 is prepared
Method is the same as the cation lipid nano particle/DNA compound being prepared in embodiment 4 with different mixing velocities Characterizing method.
Cation lipid nano particle/DNA compound average grain diameter that use device 2 is prepared is 50~150nm, PDI < 0.3, and the compound ratio about 24.82% of partial size < 150nm.
Embodiment 7 prepares cation lipid nano particle/DNA compound and its characterization using device 3:
1, cation lipid nano particle/DNA compound is prepared using device 3
(1) solution for preparing pMVA-1 (SEQ ID NO:2) and other plasmids, as pMVA-2 (SEQ ID NO:6), PMVA-3 (SEQ ID NO:7), pMVA-4 (SEQ ID NO:8) and pMVA-5 (SEQ ID NO:9), pMVA-6 (SEQ ID ) and pMVA-7 (SEQ ID NO:11) NO:10.
(2) according to shown in Fig. 2 b, it is 1 that cation lipid nano particle prepared by embodiment 1-3, which is diluted with water to concentration, After~4mg/ml, mark " cation lipid is respectively placed in mass ratio 1:1~125:1 with the plasmid of above-mentioned steps (1) preparation In nano particle " and the syringe of " DNA ", and two syringe pumps are adjusted to the same horizontal position, syringe pump are pushed, by two Cation lipid nano particle and DNA solution in a syringe are injected in t connector simultaneously with the speed of 50ml/min It is mixed, and is added dropwise in liquid storage bottle, stood 30min, obtain cation lipid nano particle/DNA compound.
(3) 0.22 μm of membrane filtration degermings.
2, cation lipid nano particle/DNA compound characterization that use device 3 is prepared
Method is the same as the cation lipid nano particle/DNA compound being prepared in embodiment 4 with different mixing velocities Characterizing method.
Cation lipid nano particle/DNA compound average grain diameter that use device 3 is prepared is 50~150nm, PDI < 0.3.
Embodiment 8, cation lipid nano particle/effect of the DNA compound to A549 cell activity
(1) plating cells culture
A549 cell in logarithmic growth phase is prepared as cell suspension, and 5 are diluted to 10%FBS-1640 × 104Cells/ml is seeded to 96 orifice plate of cell culture with 100 holes μ l/, and 37.0 DEG C, 5%CO2Under the conditions of cultivate for 24 hours.Cell is adherent Afterwards, then the culture of 1640 culture medium starvation of serum-free is replaced for 24 hours.
(2) sample to be tested is prepared
Device 1 will be utilized in embodiment 5, according to the mixture prepared DOTAP nano particle/pMVA-1 of mass ratio 10:1 Compound is diluted to 200 μ g/ml with 1640 culture mediums, then carries out 3 times of dilutions, totally 9 dilution gradients, be prepared as respectively containing The test sample of different DOTAP concentration.
(3) it is loaded
1640 culture mediums in 96 orifice plates to be inhaled and are abandoned, the test sample of step (2) is added, each gradient 3 are parallel, and totally 9 Gradient, last row is as cell blank control and blank control.37.0 DEG C, 5%CO2 cultivates 48h.
(4) CCK-8 detects cell activity
After CCK-8 and 1640 culture mediums are mixed by 1:1, added in 96 orifice plates of step (3) by 20 holes μ l/, 37.0 DEG C, After 5%CO2 cultivates 2h, OD450nm light absorption value is read in microplate reader.
(5) data are analyzed
The light absorption value and sample to be tested concentration gradient that step (4) detection obtains are fitted 4 parameter curves, in " S " curve, root Medium effective concentration (EC50) is calculated according to the curve of fitting.
As shown in figure 5, A549 tumour can be effectively suppressed in the nano particle of DOTAP prepared by the present invention/pMVA-1 compound The growth activity of cell.
Equally, cation lipid nano particle/DNA compound that prepared by embodiment 6 and embodiment 7 also has inhibition A549 The growth activity of tumour cell.
Embodiment 9, cation lipid nano particle/DNA complex stabilities test
1, the method for the present invention prepares cation lipid nano particle/DNA compound
(1), the preparation of DOTAP nano particle:
Method is the same as embodiment 1, wherein the concentration of DOTAP in ethanol is 30mg/ml, and the volume ratio of ethyl alcohol and water is 1:4。
(2), DOTAP nano particle/pMVA-1 compound preparation:
Method prepares cation lipid nano particle/DNA compound method using device 1 in embodiment 5, wherein The concentration of DOTAP nano particle is 4mg/ml, and the concentration of pMVA-1 plasmid is the 0.4mg/ml (quality of cation lipid and plasmid Than for 10:1).
2, the method for the present invention prepares cation lipid nano particle/DNA compound
(1), the preparation of DOTAP/ cholesterol (DOTAP/Chol) nano particle:
Method is the same as embodiment 2, wherein DOTAP and the concentration of cholesterol in ethanol are 30mg/ml (the two mass ratio For 1:1), the volume ratio of ethyl alcohol and water is 1:4.
(2), the preparation of DOTAP-Chol/pMVA-1 compound:
Method prepares cation lipid nano particle/DNA compound method using device 1 in embodiment 5, wherein The concentration of DOTAP/Chol nano particle is 4mg/ml, and the concentration of pMVA-1 plasmid is 0.4mg/ml (cation lipid and plasmid Mass ratio be 10:1).
3, high-pressure homogenization step is used to prepare DOTAP nano particle/DNA compound after alcohol injection
Method is the same as comparative example 1, wherein DOTAP and the concentration of cholesterol in ethanol are 10mg/ml (the two matter Amount is than being 1:1), the volume ratio of ethyl alcohol and water is 1:4；
It being formed by DOTAP/Chol-pMVA-1 compound, the concentration of DOTAP/Chol nano particle is 4mg/ml, The concentration of pMVA-1 plasmid is 0.4mg/ml (mass ratio of cation lipid and plasmid is 10:1).
3, by cation lipid nano particle/DNA compound of above-mentioned 2 kinds of distinct methods preparation (including DOTAP nanometers Grain/pMVA-1 compound, DOTAP-Chol/pMVA-1 compound) respectively 0 day and 37 DEG C accelerate after 10d detect partial size With PDI value.
The results are shown in Table 5, cation lipid nano particle/DNA compound partial size that the method for the present invention is prepared High-pressure homogenization step is used to prepare cation lipid nano particle/DNA compound partial size later less than alcohol injection, the former Uniformity be better than the latter, and accelerate after, the former stability is also superior to the latter.
In addition, the DOTAP nano particle/pMVA-1 compound and DOTAP-Chol/pMVA-1 of the method for the present invention preparation are multiple The stability for closing object is not significantly different, and shows cation lipid nano particle/DNA compound that the method for the present invention is prepared In cation lipid nano particle do not contain helper lipids when, can equally maintain the stability of compound.
Table 5. uses cation prepared by high-pressure homogenization step after comparing preparation method of the present invention and alcohol injection The partial size and PDI value of lipidic nanoparticles/DNA compound 10d in 0d and after accelerating
Equally, cation lipid nano particle/DNA compound that prepared by embodiment 6 and embodiment 7 also has good steady It is qualitative.
Embodiment 10, cation lipid nano particle/DNA compound inhibit the tumour of cervical carcinoma subcutaneous tumors model mice raw It is long
1,6-8 week old female KM mouse is raised.
2, mouse cervical cancer U14 cell culture: with 10% fetal calf serum containing inactivation, the penicillin of 100U/ml and 100 The streptomysin of μ g/ml and the DMEM culture medium of 2mM glutamine are in 37 DEG C, 5%CO2Incubator in cultivate tumour cell, Every 2 to 3 days after cell covers with sub-bottle pass on, passage control 2-10 instead of between, by logarithmic growth phase tumour it is thin Born of the same parents are used for the inoculation of in-vivo tumour.
3, tumor cell inoculation and experimental animal are grouped
Tumour cell is cleaned twice with the culture medium of antibiotic-free serum-free, is removed existing serum in cell, is then used Tumour cell is resuspended in DMEM without double antibody, and concentration is 2 × 107/ ml is subcutaneously injected, often in the right side back of experimental animal Mouse injects 100 μ l, the administration of grouping in 5 days after tumor cell inoculation, and totally 6 groups, as shown in table 6, wherein new process group refers to DOTAP nano particle/pMVA-1 compound that the present embodiment 5 is prepared (wherein the quality of DOTAP nano particle is 100 μ g, The quality of pMVA-1 plasmid is 10 μ g), old technology group refers to DOTAP nano particle/pMVA-1 that comparative example 1 is prepared Compound (it is 10 μ g that wherein the quality of DOTAP nano particle, which is the quality of 100 μ g, pMVA-1 plasmids), vehicle control group are notes What is penetrated is physiological saline.
4, close observation tumour growth situation after tumor is connect, grows to 80-100mm to tumour3When, it is tested according to table 6 to each group Mouse is administered, and administered volume is 100 μ l/.It is administered once every three days later, execution in the 21st day is all small to after connecing tumor Mouse is administered 5 times altogether, measures the gross tumor volume of every group of mouse.
The grouping situation and dosage of table 6DOTAP/pMVA-1 complex therapies cervical carcinoma subcutaneous tumors model mice
Experimental result shows the embodiment of the present invention as shown in fig. 6, the mouse volume of new process group is substantially less than other each groups The 5 DOTAP nano particle/pMVA-1 compounds being prepared can significantly inhibit the tumour growth of tumor-bearing mice, and inhibitory effect It is significantly better than DOTAP nano particle/pMVA-1 compound that old technology is prepared.
Equally, cation lipid nano particle/DNA compound that embodiment 6 and embodiment 7 are prepared also has inhibition The activity of tumor-bearing mice tumour growth.
High-pressure homogenization step is used to prepare DOTAP nano particle/DNA compound after 1. alcohol injection of comparative example
1, alcohol injection prepares cation lipid nano particle
(1) DOTAP ethanol solution is prepared:
3gDOTAP is weighed in PETG bottles of 250ml, dehydrated alcohol 100ml is added, the heating water bath in 50 DEG C gently shakes It swings to accelerate to dissolve, DOTAP ethanol solution is prepared, the concentration of DOTAP in ethanol is 30mg/ml.
(2) DOTAP ethanol solution is injected into aqueous phase solution
The good peristaltic pump of frame, setting speed be 6~8rpm, by step (1) prepare DOTAP ethanol solution be slowly added dropwise into In the aqueous solution of 400ml (volume ratio of ethyl alcohol and water is 1:4), drop outlets are maintained at 5cm or so away from liquid level.
(3) to DOTAP ethanol solution in step (2) be added drop-wise in aqueous solution complete after, dehydrated alcohol flushing pipe and PETG bottles.After the completion of flushing, continue to be dispersed with stirring system 10min with 140rpm.
2, vacuum distillation removal ethyl alcohol:
Method is the same as (4) the step of embodiment 1
3, high-pressure homogeneous whole grain
Sub-cooled circulating pump is opened, sets cryogenic temperature as 5 DEG C.With pure water rinsing high pressure homogenizer, at least 3 times.Add Enter the DOTAP nano particle that 500ml above-mentioned steps 2 are prepared, set homogeneous pressure as 500-800bar, repeatedly homogeneous 3-10 It is secondary.After the completion of homogeneous, liquid is transferred in the PETG bottle of 1L.Pure water rinsing homogenizer 3 times, then saved with dehydrated alcohol.
4, filtration sterilization: 0.22 μm of membrane filtration.
5, DOTAP nano particle/pMVA-1 compound preparation
(1) DNA solution filtration sterilization: 0.22 μm of membrane filtration pMVA-1 plasmid solution is used
(2) DOTAP/pMVA-1 compound is prepared:
In gnotobasis, by the DOTAP nano particle that above-mentioned steps 4 are prepared and the pMVA-1 that filtration sterilization obtains Plasmid solution stands 30min with the mixing of 10:1 mass ratio, forms DOTAP nano particle/pMVA-1 compound.
6, the nano particle of DOTAP prepared by high-pressure homogenization step/DNA compound characterization is used after alcohol injection
Testing result is as shown in fig. 7, use DOTAP prepared by high-pressure homogenization step nanometers after alcohol injection Grain/DNA compound average grain diameter be 150.4nm, PDI 0.238, although with this method preparation compound PDI < 0.3, But compound has polydispersity, and partial size reaches 27% (obviously beyond institute of the present invention in the compound accounting of 220nm or more Prepare compound partial size < 150nm requirement), therefore, when using 0.22 μm of membrane filtration, resistance is larger, cannot effectively reach The effect of filtration sterilization, and then this method cannot achieve large-scale production.
7, use the nano particle of DOTAP prepared by high-pressure homogenization step/DNA compound to A549 after alcohol injection The effect of cell activity
Method is the same as embodiment 8.
Testing result is as shown in Fig. 5 and table 7, although using high-pressure homogenization step (i.e. ethanol injection after alcohol injection Method+high-pressure homogeneous) prepared by DOTAP nano particle/DNA compound can inhibit the growth activity of A549 cell, but it presses down Production prepared DOTAP nano particle/DNA compound inhibiting effect low about 15.2% more of the invention.
Used after 7. the method for the present invention of table and alcohol injection DOTAP nano particle prepared by high-pressure homogenization step/ Comparison of the DNA compound to A549 cyto-inhibition
Alcohol injection+high-pressure homogeneous The method of the present invention (embodiment 5)
EC50(μg/ml) 8.879 7.709
The direct dissolution method of 2. ethyl alcohol of comparative example prepares DOTAP nano particle/DNA compound
1, DOTAP ethanol solution is prepared
DOTAP is directly dissolved in dehydrated alcohol, the ethanol solution containing 4mg/ml DOTAP is prepared as.
2, DOTAP nano particle/DNA compound is prepared
DOTAP ethanol solution prepared by above-mentioned steps 1 and the pMVA-1 plasmid solution that concentration is 0.4mg/ml are isometric Mixing (mass ratio of DOTAP and pMVA-1 are 10:1), stands 30min, forms DOTAP nano particle/pMVA-1 compound.
3, DOTAP nano particle/DNA compound characterization prepared by the direct dissolution method of ethyl alcohol
DOTAP nano particle/DNA compound average grain diameter prepared by the direct dissolution method of ethyl alcohol is 240.4nm, and PDI is 0.155。
It can be seen that DOTAP nano particle/DNA compound average grain diameter prepared by the direct dissolution method of ethyl alcohol is much larger than Compound partial size prepared by the method for the present invention.
4, compare DOTAP nano particle/DNA compound prepared by the direct dissolution method of ethyl alcohol and the method for the present invention to A549 The effect of cell activity
Method is the same as embodiment 8.
Testing result is as shown in Fig. 8 and table 8, the nano particle of DOTAP prepared by the method for the present invention/DNA compound pair The inhibiting effect of the growth activity of A549 cell is significantly higher than the inhibitory activity of compound prepared by the direct dissolution method of ethyl alcohol.
8. the method for the present invention of table and DOTAP nano particle/DNA compound prepared by the direct dissolution method of ethyl alcohol are thin to A549 The comparison of born of the same parents' inhibiting effect
5, DOTAP nano particle/DNA compound prepared by the method for the present invention and the direct dissolution method of ethyl alcohol is placed at room temperature for N Characterization after it
DOTAP nanometers prepared by DOTAP nano particle/DNA compound and comparative example 1 prepared by embodiment 5 After particle/DNA compound is placed at room temperature for 1d and 4d, the partial size and PDI of the compound that detection are prepared two methods of.
Testing result is as shown in table 9, after the compound that the method for the present invention is prepared places 1d and 4d, partial size and PDI Without significant change；And after the compound of the direct dissolution method preparation of ethyl alcohol places 4d, it is significantly increased when partial size is compared with 0d.Show this hair Cation lipid nano particle/DNA compound that bright method is prepared is distributed in unicity, partial size < 150nm, and structure is steady It is fixed, it can be with room temperature preservation.
9. the method for the present invention of table is placed at room temperature for DOTAP nano particle/DNA compound prepared by the direct dissolution method of ethyl alcohol Characterization after 1 day and 4 days
Prepared by 3. the method for the present invention of comparative example in ethanol water different final concentrations DOTAP nano particle Compound is formed by with DNA
1, the preparation of DOTAP nano particle
According to the preparation method of embodiment 1, selecting DOTAP concentration in dehydrated alcohol is 50mg/ml, ethyl alcohol and water volume Than for the preparation-obtained DOTAP nano particle of 1:5 (final concentration of the DOTAP in ethanol water is about 8.3mg/ml) (DOTAP nanoparticle sample), DOTAP concentration in dehydrated alcohol are 20mg/ml, and ethyl alcohol and water volume ratio are 1:4 (DOTAP Final concentration in ethanol water is about 4mg/ml) (the DOTAP nano particle control of preparation-obtained DOTAP nano particle Product 1) and DOTAP concentration in dehydrated alcohol be 100mg/ml, (DOTAP is water-soluble in ethyl alcohol for 1:2 for ethyl alcohol and water volume ratio Final concentration in liquid is about 33.3mg/ml) preparation-obtained DOTAP nano particle (DOTAP nano particle reference substance 2).
2, the preparation of DOTAP/pMVA-1 compound
According to the preparation method of embodiment 5, the DOTAP nano particle of various concentration is prepared in step 1, and (DOTAP is received Rice grain sample, DOTAP nano particle reference substance 1 and DOTAP nano particle reference substance 2) dilution after, with pMVA-1 plasmid according to Mass ratio 10:1 mixing, stands 30min, and it is (sample, right to be respectively formed 3 kinds of different DOTAP nano particle/pMVA-1 compounds According to product 1 and reference substance 2).
3, DOTAP nano particle/effect of the pMVA-1 compound to A549 cell activity
3 kinds of different DOTAP nano particle/pMVA-1 compounds that detecting step 2 is prepared are to A549 cell activity Effect, method is the same as embodiment 8.
Testing result is as shown in Fig. 9 and table 10, and reference substance 1 and reference substance 2 are to the inhibiting effect of A549 cell compared with sample Inhibiting effect difference low about 53.3%, 44.6%.
It can be seen that the volume ratio of final concentration of 6~25mg/ml of the DOTAP in ethanol water, ethyl alcohol and water is 1: When 3~1:6, the DOTAP nano particle that is prepared according to the method for the present invention with DNA is compound is formed by compound not only partial size It is uniform, be in monodispersity, but also with it is good inhibit growth of tumour cell biological activity.
DOTAP nano particle/pMVA-1 compound that 10 various concentration DOTAP nano particle of table and pMVA-1 plasmid are formed Comparison to A549 cyto-inhibition
Sample Reference substance 1 Reference substance 2
EC50(μg/ml) 10.25 15.72 14.82
Comparative example 4. prepares cation lipid nano particle/DNA compound in a manner of dropwise addition
1, according to the preparation method of embodiment 1, DOTAP nano particle is prepared, is diluted to 4mg/ml and is placed on In beaker and it is continuously agitated.
2, according to the method preparation preparation pMVA-1 plasmid solution of 4 step 1 of embodiment, it is diluted to 0.4mg/ml.
3, pMVA-1 plasmid solution prepared by above-mentioned steps 2 is slowly dropped to above-mentioned steps 1 with the speed of 10ml/min DOTAP nanoparticles solution in, after being added dropwise, stand 30min, to form DOTAP nano particle/pMVA-1 compound.
4, cation lipid nano particle/DNA compound characterization is prepared in a manner of dropwise addition:
Using embodiment 4 to cation lipid nano particle/DNA compound characterization detection method, the system in a manner of being added dropwise Standby obtained DOTAP nano particle/pMVA-1 compound partial size is 325.4nm, PDI=0.359.
It can be seen that long-range using cation lipid nano particle/DNA compound average grain diameter prepared by dropwise addition mode Compound partial size prepared by the method for the present invention, and the homogeneity of compound is poor (PDI > 0.3).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Chengdu Jinkai Biotechnology Co., Ltd.
<120>a kind of cation lipid nano particle/DNA compound and preparation method thereof
<130>
<160> 11
<170> PatentIn version 3.5
<210> 1
<211> 1978
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA
<400> 1
gactcttcgc gatgtacggg ccagatatac gccttctact gggcggtttt atggacagca 60
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 120
aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 180
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 240
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 300
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 360
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 420
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 480
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 540
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 600
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 660
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 720
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 780
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 840
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 900
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 960
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1020
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1080
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1140
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1200
ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1260
tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1320
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1380
taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1440
gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1500
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1560
ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 1620
ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 1680
cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 1740
aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 1800
gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 1860
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 1920
gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 1978
<210> 2
<211> 1977
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-1
<400> 2
gctgcttcgc gatgtacggg ccagatatac gccttctact gggcggtttt atggacagca 60
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 120
aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 180
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 240
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 300
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 360
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 420
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 480
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 540
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 600
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 660
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 720
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 780
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 840
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 900
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 960
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1020
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1080
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1140
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1200
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1260
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1320
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1380
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1440
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 1500
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1560
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1620
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1680
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1740
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1800
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1860
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 1920
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 1977
<210> 3
<211> 120
<212> DNA
<213> Artificial Sequence
<220>
<221> gene
<223>nucleotide sequence 1 of mtDNA
<400> 3
cccattattc ctagaaccag gcgacctgcg actccttgac gttgacaatc gagtagtact 60
cccgattgaa gcccccattc gtataataat tacatcacaa gacgtcttgc actcatgagc 120
<210> 4
<211> 600
<212> DNA
<213> Artificial Sequence
<220>
<221> gene
<223>nucleotide sequence 2 of mtDNA
<400> 4
ctgaactatc ctgcccgcca tcatcctagt cctcatcgcc ctcccatccc tacgcatcct 60
ttacataaca gacgaggtca acgatccctc ccttaccatc aaatcaattg gccaccaatg 120
gtactgaacc tacgagtaca ccgactacgg cggactaatc ttcaactcct acatacttcc 180
cccattattc ctagaaccag gcgacctgcg actccttgac gttgacaatc gagtagtact 240
cccgattgaa gcccccattc gtataataat tacatcacaa gacgtcttgc actcatgagc 300
tgtccccaca ttaggcttaa aaacagatgc aattcccgga cgtctaaacc aaaccacttt 360
caccgctaca cgaccggggg tatactacgg tcaatgctct gaaatctgtg gagcaaacca 420
cagtttcatg cccatcgtcc tagaattaat tcccctaaaa atctttgaaa tagggcccgt 480
atttacccta tagcaccccc tctaccccct ctagagccca ctgtaaagct aacttagcat 540
taacctttta agttaaagat taagagaacc aacacctctt tacagtgaaa tgccccaact 600
<210> 5
<211> 2000
<212> DNA
<213> Artificial Sequence
<220>
<221> gene
<223>nucleotide sequence 3 of mtDNA
<400> 5
tacgttgtag ctcacttcca ctatgtccta tcaataggag ctgtatttgc catcatagga 60
ggcttcattc actgatttcc cctattctca ggctacaccc tagaccaaac ctacgccaaa 120
atccatttca ctatcatatt catcggcgta aatctaactt tcttcccaca acactttctc 180
ggcctatccg gaatgccccg acgttactcg gactaccccg atgcatacac cacatgaaac 240
atcctatcat ctgtaggctc attcatttct ctaacagcag taatattaat aattttcatg 300
atttgagaag ccttcgcttc gaagcgaaaa gtcctaatag tagaagaacc ctccataaac 360
ctggagtgac tatatggatg ccccccaccc taccacacat tcgaagaacc cgtatacata 420
aaatctagac aaaaaaggaa ggaatcgaac cccccaaagc tggtttcaag ccaaccccat 480
ggcctccatg actttttcaa aaaggtatta gaaaaaccat ttcataactt tgtcaaagtt 540
aaattatagg ctaaatccta tatatcttaa tggcacatgc agcgcaagta ggtctacaag 600
acgctacttc ccctatcata gaagagctta tcacctttca tgatcacgcc ctcataatca 660
ttttccttat ctgcttccta gtcctgtatg cccttttcct aacactcaca acaaaactaa 720
ctaatactaa catctcagac gctcaggaaa tagaaaccgt ctgaactatc ctgcccgcca 780
tcatcctagt cctcatcgcc ctcccatccc tacgcatcct ttacataaca gacgaggtca 840
acgatccctc ccttaccatc aaatcaattg gccaccaatg gtactgaacc tacgagtaca 900
ccgactacgg cggactaatc ttcaactcct acatacttcc cccattattc ctagaaccag 960
gcgacctgcg actccttgac gttgacaatc gagtagtact cccgattgaa gcccccattc 1020
gtataataat tacatcacaa gacgtcttgc actcatgagc tgtccccaca ttaggcttaa 1080
aaacagatgc aattcccgga cgtctaaacc aaaccacttt caccgctaca cgaccggggg 1140
tatactacgg tcaatgctct gaaatctgtg gagcaaacca cagtttcatg cccatcgtcc 1200
tagaattaat tcccctaaaa atctttgaaa tagggcccgt atttacccta tagcaccccc 1260
tctaccccct ctagagccca ctgtaaagct aacttagcat taacctttta agttaaagat 1320
taagagaacc aacacctctt tacagtgaaa tgccccaact aaatactacc gtatggccca 1380
ccataattac ccccatactc cttacactat tcctcatcac ccaactaaaa atattaaaca 1440
caaactacca cctacctccc tcaccaaagc ccataaaaat aaaaaattat aacaaaccct 1500
gagaaccaaa atgaacgaaa atctgttcgc ttcattcatt gcccccacaa tcctaggcct 1560
acccgccgca gtactgatca ttctatttcc ccctctattg atccccacct ccaaatatct 1620
catcaacaac cgactaatca ccacccaaca atgactaatc aaactaacct caaaacaaat 1680
gataaccata cacaacacta aaggacgaac ctgatctctt atactagtat ccttaatcat 1740
ttttattgcc acaactaacc tcctcggact cctgcctcac tcatttacac caaccaccca 1800
actatctata aacctagcca tggccatccc cttatgagcg ggcgcagtga ttataggctt 1860
tcgctctaag attaaaaatg ccctagccca cttcttacca caaggcacac ctacacccct 1920
tatccccata ctagttatta tcgaaaccat cagcctactc attcaaccaa tagccctggc 1980
cgtacgccta accgctaaca 2000
<210> 6
<211> 2098
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-2
<400> 6
gactcttcgc gatgtacggg ccagatatac gccccattat tcctagaacc aggcgacctg 60
cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 120
attacatcac aagacgtctt gcactcatga gccttctact gggcggtttt atggacagca 180
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 240
aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 300
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 360
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 420
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 480
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 540
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 600
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 660
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 720
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 780
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 840
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 900
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 960
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1020
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1080
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1140
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1200
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1260
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1320
ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1380
tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1440
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1500
taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1560
gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1620
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1680
ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 1740
ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 1800
cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 1860
aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 1920
gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 1980
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2040
gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 2098
<210> 7
<211> 2578
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-3
<400> 7
gactcttcgc gatgtacggg ccagatatac gcctgaacta tcctgcccgc catcatccta 60
gtcctcatcg ccctcccatc cctacgcatc ctttacataa cagacgaggt caacgatccc 120
tcccttacca tcaaatcaat tggccaccaa tggtactgaa cctacgagta caccgactac 180
ggcggactaa tcttcaactc ctacatactt cccccattat tcctagaacc aggcgacctg 240
cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 300
attacatcac aagacgtctt gcactcatga gctgtcccca cattaggctt aaaaacagat 360
gcaattcccg gacgtctaaa ccaaaccact ttcaccgcta cacgaccggg ggtatactac 420
ggtcaatgct ctgaaatctg tggagcaaac cacagtttca tgcccatcgt cctagaatta 480
attcccctaa aaatctttga aatagggccc gtatttaccc tatagcaccc cctctacccc 540
ctctagagcc cactgtaaag ctaacttagc attaaccttt taagttaaag attaagagaa 600
ccaacacctc tttacagtga aatgccccaa ctcttctact gggcggtttt atggacagca 660
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 720
aactggatgg ctttctcgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 780
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 840
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 900
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 960
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 1020
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 1080
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 1140
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 1200
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 1260
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 1320
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 1380
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1440
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1500
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1560
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1620
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acaggtggca 1680
cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 1740
tgtatccgct catgagacaa taaccctgat aaatgcttca ataatagcac gtgctaaaac 1800
ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1860
tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1920
cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1980
taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 2040
gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 2100
acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 2160
ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2220
ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2280
cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2340
aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2400
gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2460
gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2520
gcaacgcggc ctttttacgg ttcctgggct tttgctggcc ttttgctcac atgttctt 2578
<210> 8
<211> 3978
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-4
<400> 8
gactcttcgc gatgtacggg ccagatatac gctacgttgt agctcacttc cactatgtcc 60
tatcaatagg agctgtattt gccatcatag gaggcttcat tcactgattt cccctattct 120
caggctacac cctagaccaa acctacgcca aaatccattt cactatcata ttcatcggcg 180
taaatctaac tttcttccca caacactttc tcggcctatc cggaatgccc cgacgttact 240
cggactaccc cgatgcatac accacatgaa acatcctatc atctgtaggc tcattcattt 300
ctctaacagc agtaatatta ataattttca tgatttgaga agccttcgct tcgaagcgaa 360
aagtcctaat agtagaagaa ccctccataa acctggagtg actatatgga tgccccccac 420
cctaccacac attcgaagaa cccgtataca taaaatctag acaaaaaagg aaggaatcga 480
accccccaaa gctggtttca agccaacccc atggcctcca tgactttttc aaaaaggtat 540
tagaaaaacc atttcataac tttgtcaaag ttaaattata ggctaaatcc tatatatctt 600
aatggcacat gcagcgcaag taggtctaca agacgctact tcccctatca tagaagagct 660
tatcaccttt catgatcacg ccctcataat cattttcctt atctgcttcc tagtcctgta 720
tgcccttttc ctaacactca caacaaaact aactaatact aacatctcag acgctcagga 780
aatagaaacc gtctgaacta tcctgcccgc catcatccta gtcctcatcg ccctcccatc 840
cctacgcatc ctttacataa cagacgaggt caacgatccc tcccttacca tcaaatcaat 900
tggccaccaa tggtactgaa cctacgagta caccgactac ggcggactaa tcttcaactc 960
ctacatactt cccccattat tcctagaacc aggcgacctg cgactccttg acgttgacaa 1020
tcgagtagta ctcccgattg aagcccccat tcgtataata attacatcac aagacgtctt 1080
gcactcatga gctgtcccca cattaggctt aaaaacagat gcaattcccg gacgtctaaa 1140
ccaaaccact ttcaccgcta cacgaccggg ggtatactac ggtcaatgct ctgaaatctg 1200
tggagcaaac cacagtttca tgcccatcgt cctagaatta attcccctaa aaatctttga 1260
aatagggccc gtatttaccc tatagcaccc cctctacccc ctctagagcc cactgtaaag 1320
ctaacttagc attaaccttt taagttaaag attaagagaa ccaacacctc tttacagtga 1380
aatgccccaa ctaaatacta ccgtatggcc caccataatt acccccatac tccttacact 1440
attcctcatc acccaactaa aaatattaaa cacaaactac cacctacctc cctcaccaaa 1500
gcccataaaa ataaaaaatt ataacaaacc ctgagaacca aaatgaacga aaatctgttc 1560
gcttcattca ttgcccccac aatcctaggc ctacccgccg cagtactgat cattctattt 1620
ccccctctat tgatccccac ctccaaatat ctcatcaaca accgactaat caccacccaa 1680
caatgactaa tcaaactaac ctcaaaacaa atgataacca tacacaacac taaaggacga 1740
acctgatctc ttatactagt atccttaatc atttttattg ccacaactaa cctcctcgga 1800
ctcctgcctc actcatttac accaaccacc caactatcta taaacctagc catggccatc 1860
cccttatgag cgggcgcagt gattataggc tttcgctcta agattaaaaa tgccctagcc 1920
cacttcttac cacaaggcac acctacaccc cttatcccca tactagttat tatcgaaacc 1980
atcagcctac tcattcaacc aatagccctg gccgtacgcc taaccgctaa cacttctact 2040
gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 2100
ttgggaagcc ctgcaaagta aactggatgg ctttctcgcc gccaaggatc tgatggcgca 2160
ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 2220
gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 2280
aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 2340
ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 2400
ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 2460
aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 2520
accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 2580
ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 2640
ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 2700
cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 2760
tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 2820
tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 2880
gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 2940
tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa 3000
ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt 3060
cacaccgcat acaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt 3120
tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 3180
ataatagcac gtgctaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 3240
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 3300
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 3360
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 3420
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 3480
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 3540
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 3600
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 3660
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 3720
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 3780
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 3840
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 3900
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctgggct tttgctggcc 3960
ttttgctcac atgttctt 3978
<210> 9
<211> 2097
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-5
<400> 9
gctgcttcgc gatgtacggg ccagatatac gccccattat tcctagaacc aggcgacctg 60
cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 120
attacatcac aagacgtctt gcactcatga gccttctact gggcggtttt atggacagca 180
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 240
aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 300
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 360
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 420
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 480
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 540
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 600
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 660
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 720
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 780
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 840
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 900
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 960
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1020
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1080
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1140
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1200
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1260
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1320
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1380
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1440
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1500
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 1560
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 1620
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 1680
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 1740
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 1800
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 1860
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 1920
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 1980
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2040
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 2097
<210> 10
<211> 2577
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-6
<400> 10
gctgcttcgc gatgtacggg ccagatatac gcctgaacta tcctgcccgc catcatccta 60
gtcctcatcg ccctcccatc cctacgcatc ctttacataa cagacgaggt caacgatccc 120
tcccttacca tcaaatcaat tggccaccaa tggtactgaa cctacgagta caccgactac 180
ggcggactaa tcttcaactc ctacatactt cccccattat tcctagaacc aggcgacctg 240
cgactccttg acgttgacaa tcgagtagta ctcccgattg aagcccccat tcgtataata 300
attacatcac aagacgtctt gcactcatga gctgtcccca cattaggctt aaaaacagat 360
gcaattcccg gacgtctaaa ccaaaccact ttcaccgcta cacgaccggg ggtatactac 420
ggtcaatgct ctgaaatctg tggagcaaac cacagtttca tgcccatcgt cctagaatta 480
attcccctaa aaatctttga aatagggccc gtatttaccc tatagcaccc cctctacccc 540
ctctagagcc cactgtaaag ctaacttagc attaaccttt taagttaaag attaagagaa 600
ccaacacctc tttacagtga aatgccccaa ctcttctact gggcggtttt atggacagca 660
agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta 720
aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa 780
gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg 840
gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct 900
gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac 960
ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg 1020
acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg 1080
ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa 1140
gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca 1200
ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt 1260
gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc 1320
aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc 1380
ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg 1440
ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt 1500
ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag 1560
cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttattaacgc ttacaatttc 1620
ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat caggtggcac 1680
ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 1740
gtatccgctc atgagacaat aaccctgata aatgcttcaa taatagcacg tgctaaaact 1800
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 1860
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 1920
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 1980
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 2040
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 2100
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 2160
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 2220
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 2280
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 2340
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 2400
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 2460
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 2520
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctt 2577
<210> 11
<211> 3977
<212> DNA
<213> Artificial Sequence
<220>
<221> variation
<223>nucleotide sequence of pMVA-7
<400> 11
gctgcttcgc gatgtacggg ccagatatac gctacgttgt agctcacttc cactatgtcc 60
tatcaatagg agctgtattt gccatcatag gaggcttcat tcactgattt cccctattct 120
caggctacac cctagaccaa acctacgcca aaatccattt cactatcata ttcatcggcg 180
taaatctaac tttcttccca caacactttc tcggcctatc cggaatgccc cgacgttact 240
cggactaccc cgatgcatac accacatgaa acatcctatc atctgtaggc tcattcattt 300
ctctaacagc agtaatatta ataattttca tgatttgaga agccttcgct tcgaagcgaa 360
aagtcctaat agtagaagaa ccctccataa acctggagtg actatatgga tgccccccac 420
cctaccacac attcgaagaa cccgtataca taaaatctag acaaaaaagg aaggaatcga 480
accccccaaa gctggtttca agccaacccc atggcctcca tgactttttc aaaaaggtat 540
tagaaaaacc atttcataac tttgtcaaag ttaaattata ggctaaatcc tatatatctt 600
aatggcacat gcagcgcaag taggtctaca agacgctact tcccctatca tagaagagct 660
tatcaccttt catgatcacg ccctcataat cattttcctt atctgcttcc tagtcctgta 720
tgcccttttc ctaacactca caacaaaact aactaatact aacatctcag acgctcagga 780
aatagaaacc gtctgaacta tcctgcccgc catcatccta gtcctcatcg ccctcccatc 840
cctacgcatc ctttacataa cagacgaggt caacgatccc tcccttacca tcaaatcaat 900
tggccaccaa tggtactgaa cctacgagta caccgactac ggcggactaa tcttcaactc 960
ctacatactt cccccattat tcctagaacc aggcgacctg cgactccttg acgttgacaa 1020
tcgagtagta ctcccgattg aagcccccat tcgtataata attacatcac aagacgtctt 1080
gcactcatga gctgtcccca cattaggctt aaaaacagat gcaattcccg gacgtctaaa 1140
ccaaaccact ttcaccgcta cacgaccggg ggtatactac ggtcaatgct ctgaaatctg 1200
tggagcaaac cacagtttca tgcccatcgt cctagaatta attcccctaa aaatctttga 1260
aatagggccc gtatttaccc tatagcaccc cctctacccc ctctagagcc cactgtaaag 1320
ctaacttagc attaaccttt taagttaaag attaagagaa ccaacacctc tttacagtga 1380
aatgccccaa ctaaatacta ccgtatggcc caccataatt acccccatac tccttacact 1440
attcctcatc acccaactaa aaatattaaa cacaaactac cacctacctc cctcaccaaa 1500
gcccataaaa ataaaaaatt ataacaaacc ctgagaacca aaatgaacga aaatctgttc 1560
gcttcattca ttgcccccac aatcctaggc ctacccgccg cagtactgat cattctattt 1620
ccccctctat tgatccccac ctccaaatat ctcatcaaca accgactaat caccacccaa 1680
caatgactaa tcaaactaac ctcaaaacaa atgataacca tacacaacac taaaggacga 1740
acctgatctc ttatactagt atccttaatc atttttattg ccacaactaa cctcctcgga 1800
ctcctgcctc actcatttac accaaccacc caactatcta taaacctagc catggccatc 1860
cccttatgag cgggcgcagt gattataggc tttcgctcta agattaaaaa tgccctagcc 1920
cacttcttac cacaaggcac acctacaccc cttatcccca tactagttat tatcgaaacc 1980
atcagcctac tcattcaacc aatagccctg gccgtacgcc taaccgctaa cacttctact 2040
gggcggtttt atggacagca agcgaaccgg aattgccagc tggggcgccc tctggtaagg 2100
ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc gccaaggatc tgatggcgca 2160
ggggatcaag ctctgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg 2220
gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac 2280
aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg 2340
ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc 2400
ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg 2460
aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc 2520
accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc 2580
ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta 2640
ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg 2700
cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg 2760
tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat 2820
tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc 2880
gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta 2940
tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa 3000
ttattaacgc ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt 3060
cacaccgcat caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt 3120
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa 3180
taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 3240
gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 3300
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 3360
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 3420
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 3480
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 3540
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 3600
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 3660
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 3720
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 3780
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 3840
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 3900
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 3960
tttgctcaca tgttctt 3977

Claims (12)

1. a kind of preparation method of cation lipid nano particle/DNA compound, which is characterized in that the method includes following Step:
(1) cationic lipid material is dissolved in dehydrated alcohol, is dissolved by heating；
(2) ethanol solution containing cationic lipid material that above-mentioned steps (1) are prepared is added drop-wise in aqueous phase solution, from Assembling forms cation lipid nano particle；
(3) remaining ethyl alcohol in above-mentioned steps (2) cation lipid nano particle is removed；
(4) it filters；
(5) DNA solution is prepared；
(6) the cation lipid nano particle and above-mentioned steps that above-mentioned steps (4) are prepared are mixed according to certain mass ratio (5) DNA solution being prepared, to form cation lipid nano particle/DNA compound；
(7) it filters；
Wherein, the concentration of cationic lipid material in ethanol described in step (1) is 30~100mg/ml, preferably 50- 60mg/ml；The volume ratio of ethanol solution liquid compatible with water described in step (2) is 1:3~1:6, the cation lipid Final concentration of 6~25mg/ml of the material in ethyl alcohol and water phase mixed solution；
Formed cation lipid nano particle/DNA compound partial size is 50~150nm, PDI < 0.3.
2. preparation method according to claim 1, which is characterized in that cationic lipid material described in step (1) is Cation lipid is selected from (2,3-Dipropyloxypropyl)trimethylammonium chloride (DOTAP), N- [1- (2,3- dioleoyl chlorine) third Base]-N, N, oily alkenyloxy group propyl -2- (the 2- spermine formyl ammonia of N- trimethyl ammonium chloride (DOTMA), trifluoroacetic acid dimethyl -2,3- two Base) ethyl ammonium (DOSPA), bromination trimethyldodecane base ammonium (DTAB), Tetradonium Bromide (TTAB), bromination three At least one of methyl cetyltrimethyl ammonium (CTAB) or bromoethyl dibasecylammonium bromide (DDAB).
3. preparation method according to claim 2, which is characterized in that the cationic lipid material is the cationic lipid The mixture of matter and helper lipids, wherein the mass ratio > 1:1 of cation lipid and helper lipids, the helper lipids are selected from In phosphatidyl-ethanolamine (PE), phosphatidyl choline (PC), cholesterol (Chol) or dioleoylphosphatidylethanolamine (DOPE) It is at least one.
4. preparation method according to claim 1-3, which is characterized in that cationic lipid described in step (6) Matter nano particle and the DNA mass ratio are 6:1~125:1.
5. preparation method according to claim 1, which is characterized in that cation lipid nanometer described in step (6) Grain with the DNA solution be prepared as in the semiautomatic plant containing t connector described in cation lipid nano particle/ DNA compound；The construction of the semiautomatic plant of the t connector is according to arrow direction by constant flow pump, sterile filters, storage Liquid bottle, constant flow pump, t connector are connected in sequence, first semiautomatic plant of the assembling containing t connector, then will step Suddenly the DNA solution that the cation lipid nano particle and step (5) that (4) are prepared are prepared is with the quality of 6:1~125:1 Than being respectively placed in the container of described device, it is added newly in t connector with stream after the speed mixing of 20~100ml/min In container.
6. preparation method according to claim 1, which is characterized in that cation lipid nanometer described in step (6) Grain is that the cation lipid nano particle that above-mentioned steps (4) are prepared is obtained after water dilutes, and the concentration after dilution is 1 ~4mg/ml.
7. preparation method according to claim 1, which is characterized in that heating is heating water bath, water-bath described in step (1) The temperature of heating is 40~60 DEG C, preferably 50~60 DEG C.
8. preparation method according to claim 1, which is characterized in that aqueous phase solution described in step (2) is aqueous solution, Or the aqueous solution containing glucide, glucide are selected from lactose, maltose, sucrose, glucose or trehalose, glucide Mass concentration is 2-20%, preferably 4-10%.
9. preparation method according to claim 1, which is characterized in that rate of addition described in step (2) be 30~ 120ml/min；The method of ethyl alcohol is removed described in step (3) in revolving, dialysis, ultrafiltration, spray drying or freeze-drying It is a kind of；Filtering is using 0.22 μm of membrane filtration described in step (4) and step (6).
10. preparation method according to claim 1, which is characterized in that DNA described in step (5) is plasmid, plasmid choosing From at least one of pVAX1, pcDNA3.1, pBR322 or pUC18；Or the plasmid is pMVA plasmid, nucleotides sequence Column as shown in SEQ ID NO:1 or its nucleotide sequence and sequence shown in SEQ ID NO.1 have 90% or more it is homologous Property；Or the plasmid is pMVA-1 plasmid, the nucleotides sequence of the pMVA-1 plasmid is classified as shown in SEQ ID NO.2, or Its nucleotide sequence has 90% or more homology with sequence shown in SEQ ID NO.2；Or the plasmid is selected from nucleotide Sequence such as SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10 or SEQ ID Shown in NO.11 or nucleotide sequence and SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ Sequence shown in ID NO.10 or SEQ ID NO.11 has at least one of 90% or more plasmid of homology；
Or DNA described in step (5) is mitochondrial DNA or mitochondria DNA fragment, mitochondria DNA fragment is selected from nucleotide Sequence is DNA fragmentation or nucleotide sequence and SEQ shown in SEQ ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 Sequence shown in ID NO.3, SEQ ID NO.4 or SEQ ID NO.5 has at least one in the DNA fragmentation of 90% or more homology Kind.
11. preparation method according to claim 1, which is characterized in that DNA solution described in step (5) is DNA water-soluble Liquid, the length of DNA are 100~2500bp.
12. a kind of cation lipid nano particle/DNA compound, which is characterized in that the compound passes through claim 1-11 Described in any item preparation methods are prepared.
CN201810038987.4A 2017-12-11 2018-01-16 Cationic lipid nanoparticle/DNA compound and preparation method thereof Active CN109893664B (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201711308308 2017-12-11
CN2017113083082 2017-12-11

Publications (2)

Publication Number Publication Date
CN109893664A true CN109893664A (en) 2019-06-18
CN109893664B CN109893664B (en) 2022-03-11

Family

ID=66943214

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810038987.4A Active CN109893664B (en) 2017-12-11 2018-01-16 Cationic lipid nanoparticle/DNA compound and preparation method thereof

Country Status (1)

Country Link
CN (1) CN109893664B (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109125740A (en) * 2017-06-28 2019-01-04 四川大学 A kind of novel tumor vaccine and application thereof
CN110974954A (en) * 2019-12-24 2020-04-10 珠海丽凡达生物技术有限公司 Lipid nanoparticle for enhancing immune effect of nucleic acid vaccine and preparation method thereof
CN111926077A (en) * 2020-06-29 2020-11-13 江苏省人民医院（南京医科大学第一附属医院） Application of mitochondria-derived mc-COX2 as potential marker in CLL
CN112111483A (en) * 2020-09-16 2020-12-22 南通大学 dsDNA microwave melting method for keeping bacterial activity
CN112999351A (en) * 2021-03-11 2021-06-22 华中农业大学 Preparation method and application of artificial lipid drops and freeze-dried preparation thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040142025A1 (en) * 2002-06-28 2004-07-22 Protiva Biotherapeutics Ltd. Liposomal apparatus and manufacturing methods
CN106474064A (en) * 2015-08-27 2017-03-08 昆药集团股份有限公司 A kind of Artemether nanometer liposome and preparation method and application

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040142025A1 (en) * 2002-06-28 2004-07-22 Protiva Biotherapeutics Ltd. Liposomal apparatus and manufacturing methods
CN106474064A (en) * 2015-08-27 2017-03-08 昆药集团股份有限公司 A kind of Artemether nanometer liposome and preparation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YOSHIE MAITANI ET AL.: "Cationic liposome (DC-Chol/DOPE = 1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression", 《INTERNATIONAL JOURNAL OF PHARMACEUTICS》 *
魏霞蔚1，张志荣: "用作基因传递系统的阳离子纳米载体的细胞毒性及其机制研究进展", 《药学进展》 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109125740A (en) * 2017-06-28 2019-01-04 四川大学 A kind of novel tumor vaccine and application thereof
CN110974954A (en) * 2019-12-24 2020-04-10 珠海丽凡达生物技术有限公司 Lipid nanoparticle for enhancing immune effect of nucleic acid vaccine and preparation method thereof
CN111926077A (en) * 2020-06-29 2020-11-13 江苏省人民医院（南京医科大学第一附属医院） Application of mitochondria-derived mc-COX2 as potential marker in CLL
CN112111483A (en) * 2020-09-16 2020-12-22 南通大学 dsDNA microwave melting method for keeping bacterial activity
CN112999351A (en) * 2021-03-11 2021-06-22 华中农业大学 Preparation method and application of artificial lipid drops and freeze-dried preparation thereof
CN112999351B (en) * 2021-03-11 2022-06-10 华中农业大学 Preparation method and application of artificial lipid drops and freeze-dried preparation thereof

Also Published As

Publication number Publication date
CN109893664B (en) 2022-03-11

Similar Documents

Publication Publication Date Title
CN109893664B (en) Cationic lipid nanoparticle/DNA compound and preparation method thereof
US11738072B2 (en) Tumor vaccine and uses thereof
US20210040472A1 (en) Methods and apparatuses for manufacturing for removing material from a therapeutic composition
CN113073096B (en) Method and reagent for separating and enriching pathogenic microorganisms in blood and extracting nucleic acid
CN100542614C (en) The virus envelope vector that is used for gene transfer
CN101873867B (en) The compositions of high concentration chitosan-nucleic acid complexes
CN109640946A (en) The negative-feedback regu- lation of HIV-1 is carried out by gene editing strategy
CN107312799B (en) Lentiviral vector cryopreservation protective solution and preparation method and application thereof
JP2019524148A (en) Microbial concentration or nucleic acid extraction method using DTBP
CN104774270B (en) A kind of gland cancer specificity EpCAM GM CSF gene recombinant fusion proteins and preparation method thereof
US20170334949A1 (en) Reagent kit and method for removing bacterial endotoxin in biological product
CN115778904A (en) A preparation for in vitro transfection and in vivo mRNA delivery
Carvalho et al. Bioorthogonal strategy for bioprocessing of specific-site-functionalized enveloped influenza-virus-like particles
CN115029320B (en) Engineering exosome for tumor radiotherapy sensitization, preparation method and application
US20190117572A1 (en) Microfluidic-formulated leukosome compositions and fabrication methods therefor
CN115252582A (en) Preparation and application of erythrocyte membrane heterozygosis pH liposome coated oncolytic virus preparation
CN108330135A (en) Modify fusion, the plasmid with the fusion, lentiviral particle, stem cell and the application of mescenchymal stem cell
CN112424346A (en) Solubilized adenosine triphosphatase, methods and uses
CN109701004A (en) The method for removing pollutant
RU2539030C1 (en) Kit for dna recovery
CN110423752A (en) SiRNA, recombinant vector, cell model and the application in anti-tumor drug of silencing Ku70 gene
CN112029802A (en) Preparation method and application of exosome rich in human keratinocyte growth factor-2
NL2027654B1 (en) Methods for manufacturing a syntetic template
BR102015013040A2 (en) process of obtaining outer membrane vesicles (omv) from the haemophilus aegyptius 254 (hae254) strain, outer membrane vesicles thus obtained (omv-hae254) and their use for cell transfection
Sobotkova et al. Characteristic of two mouse bcr-abl-transformed cell lines: I. General properties of the cells

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
CB02 Change of applicant information

Address after: 610041 South Keyuan Road, Chengdu High-tech Zone, Chengdu, Sichuan Province, No. 88, 10 buildings, 2 floors, 203

Applicant after: Xinlitai (Chengdu) Biotechnology Co., Ltd.

Address before: 610041 South Keyuan Road, Chengdu High-tech Zone, Chengdu, Sichuan Province, No. 88, 10 buildings, 2 floors, 203

Applicant before: Chengdu Jinkai Biological Technology Co., Ltd.

CB02 Change of applicant information
TA01 Transfer of patent application right

Effective date of registration: 20191101

Address after: 610041, No. 2, No. 88, 10, South Garden Road, Chengdu hi tech Zone, Sichuan, Chengdu, 203

Applicant after: Xinlitai (Chengdu) Biotechnology Co., Ltd.

Applicant after: Shenzhen Xinlitai Pharmaceutical Co., Ltd.

Address before: 610041, No. 2, No. 88, 10, South Garden Road, Chengdu hi tech Zone, Sichuan, Chengdu, 203

Applicant before: Xinlitai (Chengdu) Biotechnology Co., Ltd.

TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20201201

Address after: 610041, No. 24, south section of first ring road, Chengdu, Sichuan, Wuhou District

Applicant after: SICHUAN University

Address before: 610041, No. 2, No. 88, 10, South Garden Road, Chengdu hi tech Zone, Sichuan, Chengdu, 203

Applicant before: Xinlitai (Chengdu) Biotechnology Co.,Ltd.

Applicant before: SHENZHEN SALUBRIS PHARMACEUTICALS Co.,Ltd.

TA01 Transfer of patent application right
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant