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.
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