CN108893488A - A kind of recombinant plasmid, DNA vaccination and its preparation method and application - Google Patents
A kind of recombinant plasmid, DNA vaccination and its preparation method and application Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Abstract
The invention discloses the recombinant plasmids of specific treatment IBD a kind of, DNA vaccination and its preparation method and application.In experimentation of the invention, the Injured colonic mucosa degree of the IBD model mice of the treatment group through DNA vaccination administration is substantially reduced, and the scoring of enteritis activity index is lower than model group, and the proinflammatory inflammation factor TNF-α of model group enteron aisle, IL-6 increase, CD4+CD25+Foxp3+T cell be lower than pVAX1-TNFAIP3 treatment group.Meanwhile the phosphorylation level for the treatment of group's GAP-associated protein GAP is substantially less than model group, shows that pVAX1-TNFAIP3 can inhibit the generation of inflammation by the activation of inhibition related inflammation signal path；In addition, colon Foxp3 protein upregulation, shows that pVAX1-TNFAIP3 can inhibit the generation of inflammation by the increase of up-regulation regulatory T cells.These all confirm that pVAX1-TNFAIP3 recombinant plasmid and DNA vaccination obtained have good therapeutic effect to inflammatory bowel disease.
Description
Technical field
The present invention relates to genetic engineering field, in particular to a kind of recombinant plasmid, DNA vaccination and preparation method thereof and answer
With.
Background technique
Inflammatory bowel disease (inflammatory bowel disease, IBD) is a kind of common chronic nonspecific enteron aisle
Disease is divided into two classes according to clinical and Pathologic Characteristics：Ulcerative colitis (ulcerativecolitis, UC) and Crow grace
Sick (Crohn ' s disease, CD).In recent years, with the pollution of the raising of living standards of the people and environment, IBD disease incidence and
Illness rate, in the trend that rises appreciably, seriously affects minimal invasive treatment and work in China, carrys out great burden to society, economy-zone.IBD
The cause of disease be not yet illustrated at present, it is considered that have with genetic predisposition, immune inflammation, intestinal flora and psychologic factors etc.
It closes.Wherein, the key of pathogenesis may be immune system for the reaction of the abnormal immune of intestinal flora and food antigens, cause
A large amount of inflammatory factor inflammation damnification caused by enteron aisle localized clusters.
IBD current therapeutic agent includes Salicylic Acid Formulations, glucocorticoid and some immunosuppressant drugs, but is lacked
The treatment means of weary specificity, therefore find new treatment means and have become the hot and difficult issue studied both at home and abroad.
Summary of the invention
The object of the present invention is to provide the recombinant plasmid of specific treatment IBD a kind of, DNA vaccination and preparation method thereof and answer
With.
To achieve the above object, the technical solution adopted by the present invention is that：
A kind of recombinant plasmid, including TNFAIP3 gene and eukaryotic expression vector pVAX1, the TNFAIP3 gene
GenBank accession number is KJ892292.1.
The preparation method of above-mentioned recombinant plasmid, specific step is as follows：Segment comprising TNFAIP3 gene is inserted into carrier
Between EcoR I and EcoR the V restriction enzyme site of pVAX1.
Application of the above-mentioned recombinant plasmid in the drug that preparation prevents and treats inflammatory bowel disease.
A kind of DNA vaccination, including above-mentioned recombinant plasmid.
Preferably, which is multi-phase emulsion.
It preferably, further include poly lactide-glycolide acid, polyvinyl alcohol.
A kind of preparation method of DNA vaccination, specific step is as follows：
It takes poly lactide-glycolide acid to be dissolved in organic solvent, forms organic phase；
The solution for taking above-mentioned recombinant plasmid is mixed to form lotion with the organic phase；
The lotion is mixed with polyvinyl alcohol water solution, forms multi-phase emulsion.
The W1/O/W2 type multi-phase emulsion obtained by the preparation method is used directly for colon administration, directly acts on
IBD pathogenic site is not necessarily to intramuscular injection.
Preferably, which is added deionized water, and after organic solvent volatilization, precipitating is collected in centrifugation.
DNA vaccination made from the above method.
Above-mentioned DNA vaccination prevents and treats the application in inflammatory bowel disease in preparation.
The beneficial effects of the invention are as follows：
The Injured colonic mucosa degree for the IBD model mice being administered through DNA vaccination of the invention is substantially reduced, enteritis
Activity index scoring is lower than model group, shows that pVAX1-TNFAIP3 has preventive and therapeutic action to the colitis of induction.Model group
The proinflammatory inflammation factor TNF-α of enteron aisle, IL-6 increase, CD4+CD25+Foxp3+T cell be lower than pVAX1-TNFAIP3 intervention group.Together
When, the phosphorylation level of intervention group GAP-associated protein GAP is substantially less than model group, shows that pVAX1-TNFAIP3 can be by inhibiting related
The activation of inflammatory signals access inhibits the generation of inflammation；In addition, colon Foxp3 protein upregulation, shows pVAX1-TNFAIP3
It can inhibit the generation of inflammation by the increase of up-regulation regulatory T cells.These all confirm pVAX1-TNFAIP3 to inflammatory bowel
Disease has good therapeutic effect.
Detailed description of the invention
Fig. 1 is the building of IBD model mice and administration time arrangement figure in one embodiment of the present of invention.
Fig. 2 is the external plasmid construction of pVAX1-TNFAIP3 and nano-packaging result figure in the embodiment of Fig. 1 of the invention.
Wherein Figure 1A is double digestion as a result, Electronic Speculum result, Fig. 1 D (I) and Fig. 1 D that Figure 1B is sequencing result, Fig. 1 C is nano-packaging
It (II) is the result of in-vitro transfection 293T cell.
Fig. 3 is control group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls mouse
Changes of weight figure.
Fig. 4 is control group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls mouse
DAI appraisal result.
Fig. 5 is the mouse Colon comparison diagram in the embodiment of Fig. 1 of the invention, is from left to right control group, model respectively
Group, intervention group, unloaded group and positive controls.
Fig. 6 is the HE colored graph (200 ×) in the embodiment of Fig. 1 of the invention, and 5A, 5B, 5C, 5D, 5E are respectively represented pair
According to group, model group, intervention group, unloaded group and positive controls.
Fig. 7 is control group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls mouse
Colon IL-4, IL-6, IFN-γ and TIM1 expression figure.
Fig. 8 is normal group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls mouse
Spleen monocyte in CD4+CD25+Foxp3+T cell ratio chart；
Fig. 9 is normal group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls mouse
Inflammatory signals pathway protein expression figure in colonic tissue.
Figure 10 is that normal group in the embodiment of Fig. 1 of the invention, model group, intervention group, unloaded group and positive controls are small
Foxp3 protein expression situation map in mouse colonic tissue.
Specific embodiment
It is clearly and completely described below with reference to technical effect of the embodiment to design and generation of the invention, with
It is completely understood by the purpose of the present invention, feature and effect.
Embodiment 1
1. experimental method
Experimental animal and grouping
Female is selected, 6-8 week old, is purchased from Guangdong Medical Lab Animal Center by C57BL/6 healthy mice 30.At random
It is divided into 5 groups, every group 6, is divided into control group, model group, intervention group, unloaded group and positive controls, the place of every group of experimental animal
Reason mode is as shown in table 1.It is recorded twice weight weekly, stool in mice is observed, for judging mouse DIA index；Weekly the 7th
Its (namely 7,14,21,42,49 days) collect excrement for detecting experiment of occulting blood；Last eyeball of mouse takes blood, for detecting phase
The inflammatory factor of pass, mouse Colon is extracted for RNA, Protein Extraction and HE dyeing, this experiment use dextran sulfate sodium
(Dextran sulfate sodium salt, DSS) (article No.：216011080) modeling is carried out to mouse, specific dosage is timely
Between arrange see Fig. 1.
The grouping of 1. experimental animal of table and disposition
The building of recombinant plasmid (pVAX1-TNFAIP3 carrier for expression of eukaryon)
Expand TNFAIP3 (GenBank：KJ892292.1 gene coded sequence) passes through the restriction enzyme site of EcoR I
(GAATTC) it is building up on pVAX1 carrier with the restriction enzyme site of EcoR V (GATATC), finally by screening, extracts plasmid and send
Sequencing company sequencing, verifies pVAX1-TNFAIP3 carrier for expression of eukaryon accuracy；The gene for expanding mCherry-TNFAIP3 is compiled
Code sequence, and be building up in carrier by the restriction enzyme site (GAATTC) of EcoR I and the restriction enzyme site (TCTAGA) of Xbal to build
Vertical pVAX1-mcherry-TNFAIP3 carrier for expression of eukaryon extracts plasmid and sequencing company is sent to be sequenced, test finally by screening
Demonstrate,prove pVAX1-mcherry-TNFAIP3 carrier for expression of eukaryon accuracy.Before rectum is to 16h, it is copolymerized with poly lactic-co-glycolic acid
Object and polyvinyl alcohol are to contain material, using supersound method, prepare pVAX1-TNFAIP3 and pVAX1-mcherry-
TNFAIP3 nanoparticle；Wherein for pVAX1-TNFAIP3 for treating, pVAX1-mcherry-TNFAIP3 is used for Validation in vitro
TNFAIP3 albumen can express in the cell.The external plasmid construction result of pVAX1-TNFAIP3 is shown in Fig. 2.
Primer is according to (GenBank：KJ892292.1 gene coded sequence design), the raw work bioengineering in committee Shanghai are limited
Company's synthesis, specific primer information are seen below：
TNFAIP3 amplimer：
Upstream primer SEQ NO.2：CGGAATTCCACCATGGCTGAACAAGT；
Downstream primer SEQ NO.3：CCGGATATCTTA GCCATACATCTGC.
MCherry-TNFAIP3 amplimer：
Upstream primer SEQ NO.4：CGGAATTCCACCATGGCTGAACAAGT；
Downstream primer SEQ NO.5：GCTCTAGAGCTTACTTGTACAGCTCG.
CDS sequence in pVAX1-TNFAIP3 and pVAX1-mcherry-TNFAIP3 building process is respectively such as SEQ
NO.6-7。
The preparation of pVAX1-TNFAIP3 DNA vaccination
It takes the poly lactide-glycolide acid of 100mg to be dissolved in 900 μ L methylene chloride and 100 μ L acetone, is formed organic
Phase；10 μ L are taken to be dissolved in the pVAX1-TNFAIP3 plasmid (solution concentration is 1 μ g/ μ L) of sterile ultrapure water, as inner aqueous phase；To have
Machine phase and inner aqueous phase two-phase mixtures, in ice-water bath, ultrasonication 20 (power 40W, intermittent times/stream time
For 6s/6s), lotion is formed；In lotion inject 2mL 2% polyvinyl alcohol water solution be used as outer aqueous phase, again ultrasound (power
For 40W, intermittent time/stream time is 6s/6s), form multiphase emulsion (can in stirring side be added).In multinomial emulsion
Middle addition 50mL deionized water, is stirred at room temperature (or rotary evaporation) 4h and volatilizees completely to organic solvent；It is carried out under the conditions of 4 DEG C
20min is centrifuged (revolving speed 10000rpm), collects precipitating, sterile washing 3 times；Freeze-drying, -80 DEG C of preservations, for use.pVAX1-
The nano-packaging of TNFAIP3 plasmid is shown in Fig. 2.
The preparation of IBD model
Control group mice drinks sterile water, other four groups in addition to control group were dissolved in 1,4,7 week with 2%DSS sterile
After water, free water.It -3rd, 4,18,24,39,45 day, is filled by enema syringe into corresponding drug (intervention group：pVAX1-
20 μ of TNFAIP3 g/, unloaded group：20 μ of pVAX1 g/, positive controls：By 62.5mg/kg medication), administration time arranges table
See Fig. 1.PVAX1-TNFAIP3 used in model preparation can be the multi-phase emulsion during being prepared as above, can be by the cream
Liquid is directly used in colon administration, directly acts on IBD pathogenic site, is not necessarily to intramuscular injection.Also the nanometer after precipitating can be used
Particle carries out colon administration, carries out specific treatment.
2. experimental result
Model construction situation
The weight of five groups of mouse is measured respectively, situation of change is as shown in Figure 3.Control group mice stool and urine is just
Often, weight gain, hair is glossy, diet, activity, the state of mind are normal.Compared to the mouse of control group, model group mouse
Then there is mucus loose stools at beginning the 3rd day or so of medication in 1,4,7 week, and symptom gradually aggravates, symptom is more tight within 5 days or so
Weight, it is seen that symptoms, the intervention group such as pus and blood stool, syntexis, weight loss, hair are matt, diet significantly reduces, chilly, lazy move are small
Mouse started mucus loose stools occur at 5 days or so, and gradually significantly reduced with syntexis, weight loss, hair tarnish, diet,
The symptoms such as chilly, lazy move, while mucus loose stools switchs to pus and blood stool.Pus and blood stool, weight loss, diet significantly reduce, chilly, lazy move
Deng the result shows that the apparent colitis of model group appearance, shows that IBD model is successfully established.
Fecal occult blood experiment
It occults blood experimental grade criterion：Negative (-)：Blue-green is not shown after 3 minutes；Weakly positive (+)：Indigo plant is shown in 30-60 seconds
Color；Positive (++)：Blue-green is shown immediately；Strong positive (+++)：Navy blue is shown immediately.
Fecal occult blood experiment is carried out to five groups of mouse, experimental result display model group shows navy blue immediately, shows as strong sun
Property, and intervention group shows blue-green after 30 seconds, shows as weakly positive.The result shows that compared with the control group, model group and zero load
Group stool in mice is occulted blood seriously, and intervention group and positive controls mouse in process pVAX1-TNFAIP3 and
The symptoms were significantly improved for fecal occult blood after Sulfasalazine intervenes.
Mouse DAI scoring
It is tested in conjunction with above-mentioned fecal occult blood, colitis disease activity index (disease activity index, DAI) is commented
The standard with reference to Suthceland LR is divided to carry out, specific standards of grading are：Shaped particles shape class just, occults blood and tests negative scoring
0；The half graininess class for being loosely not adhere to anus just, occult blood and test score positive 2 by negative scoring 1 of such as occulting blood；It is adhered to anus
Liquid state just, occult blood and test score positive 3, naked eyes visible bloody stool scoring 4.
Five groups of mouse colitis injury severity scores are assessed, calculate the DAI of 5 groups of mouse weekly, as a result such as Fig. 4 institute
Show.The disease activity index scoring of model group is significantly higher than control group (P<0.01), and intervention group and positive controls are significantly low
In model group (P<0.01).The start within 3-5 days to occur lazy move, loose stools, glutinous after modeling with the model group mouse of weight loss
Liquid bloody stool, and the DAI of intervention group is reduced compared with model group, intervention group weight changes compared with model group indifference, the disease of intervention group mouse
Shape improves obviously, this shows that pVAX1-TNFAIP3 can be obviously improved the colitis damage of mouse.
Mouse Colon observation
At the 49th day, cervical dislocation put to death five groups of mouse, is rounded section colon, substantially carries out shape to the colon of three groups of mouse
State observation, as a result as shown in Figure 5.Experimental result display model group mouse Colon significantly shortens, it is seen that obvious congestion and edema has
Large area is rotten to the corn.Compared with model group, intervention group colon oedema is lighter, and rotten to the corn area is small, shows that pVAX1-TNFAIP3 has
Certain treatment curative effect.
HE dyeing
Five groups of mouse Colon segmental colonic tissues are taken, PBS is rinsed well, fixes 24 hours with formic acid solution.
1) dehydration with it is transparent
Dehydration is gradually carried out to high concentration ethanol from low-concentration ethanol.Sample is first placed in the of short duration guarantor of 70% ethyl alcohol
It deposits, can both be dehydrated, while also playing the role of continuing fixation to sample.When starting film-making, after sample is stayed overnight in 80% ethyl alcohol, according to
Secondary immersion volume fraction is 95% ethyl alcohol I, II each 2h, 100% ethyl alcohol I, II each 1.5h.Dimethylbenzene I, II is transparent twice, respectively
40min/ times.Detect by an unaided eye transparency in clearing process, avoids transparent insufficient or excessive.
2) embedding and slice
I 30min of paraffin, II 1h of paraffin, III 1.5h of paraffin, conventional waxdip embedding (58-60 DEG C of wax fusing point of embedding) cut 5 μ
M thickness wax disk(-sc), 42 DEG C of water-baths are interior to open up piece, conventional roasting piece.
3) it dyes
Yihong hematoxylin ﹣ (HE) dyeing：Baked wax disk(-sc) is dewaxed each 10min through dimethylbenzene I, II, dehydrated alcohol, 95%
Gradually rehydration is to 70% ethyl alcohol for ethyl alcohol, and every grade of 2min, into after distilled water 3min, hematoxylin dyes 15min, 1% hydrochloride alcohol point
Change 30s, flowing water rinses 20min, eosin stains 2min, crosses water, 95% ethyl alcohol 2min, 100% ethyl alcohol 2min, dimethylbenzene I, II is respectively
5min, neutral gum mounting.
Histological score standard:1. inflammation：Nothing, 0 point；Slightly, 1 point, moderate, 2 points；Severe, 3 points.2. goblet cell：Not
See disappearance, 0 point；It disappears, 1 point.3. injured depth：Nothing, 0 point；Submucosa, 1 point；Muscle layer, 2 points；Placenta percreta, 3 points.4. bursting
Ulcer：Nothing, 0 point；Erosion, 1 point；Ulcer, 2 points.5. crypt abscess：Nothing, 0 point；Have, 1 point.
Referring to Fig. 6, control group and positive controls mouse Colon mucous membrane are completely continuous, body of gland marshalling, and no oedema is burst
Ulcer and rotten to the corn formation.And the mouse Colon mucosa injury of model group and unloaded group is heavier, it is seen that obvious congestion and edema, extensively big face
Long-pending erosion, deep layer ulcer are formed, body of gland be destroyed it is disorganized, mucous membrane and the visible big amount lymphocyte of submucosa, it is thermophilic in
Property granulocyte infiltration.Mucosa injury degree is substantially reduced under intervention group mouse mirror, and colonic mucosa is complete, partially visible mild hyperaemia
Oedema and a small amount of inflammatory cell infiltration show that pVAX1-TNFAIP3 has apparent treatment curative effect.
Inverse transcription polymerase chain reaction
Mouse nasal mucosal tissue total serum IgE (n=3) is extracted using RNeasy Mini Kit kit, ultraviolet specrophotometer
Measure the concentration and purity of RNA.According to II 1st strand cDNA Synthesis kit reagent of TaKaRa PrimeScript
2 μ g RNA reverse transcriptions are cDNA by box specification, and reacting whole system is 20 μ l.Use TaKaRa SYBR Premix Ex Taq
II kit carries out RT-PCR amplification by ABI 7500, and the amplification first step is：95 DEG C, 30s；It is followed by 95 DEG C, 3s and 60 DEG C,
40 circulations of 30s are used as second step.Using 5.0 software design qPCR primer of Primer, with IL-4, IL-6, IFN-γ,
Gene, actin use 2 as internal reference to TIM1 as a purpose-ΔΔCtThe relative expression quantity of method calculating gene.Model as the result is shown
Expression of the group compared to intervention group and positive controls IL-4, IL-6, TIM1 increases (see Fig. 7), and intervention group is compared to the positive
The expression of control group IL-4, IL-6, TIM1 are less.Show that pVAX1-TNFAIP3 has certain therapeutic effect.
Flow cytometry CD4+CD25+Foxp3+The ratio of T cell
Disconnected neck puts to death mouse, and sterile taking-up mouse spleen (n=6) puts it into and is marked with mouse lymphocyte separating liquid
In culture dish (35mm), spleen monocyte is prepared according to specification, cell concentration is diluted to 1 × 106/mL.First use APC Rat
Anti-Mouse CD4 and FITC Rat Anti-Mouse CD25 streaming antibody dyes 15min, then with how long formaldehyde is fixed
15min, PBS are washed three times, are finally worn film liquid stained over night with containing PE Rat Anti-MouseFoxp3, are used streaming within second day
Cell instrument detects CD4 in spleen monocyte+CD25+Foxp3+The ratio of T cell, and data are divided using Flowjo software
Analysis.Intervention group and positive controls are compared with model group and unloaded group CD4 as the result is shown+CD25+Foxp3+T cell ratio increases (see figure
8), and intervention group is higher compared to positive controls CD4+CD25+Foxp3+T cell proportion, due to CD4+CD25+Foxp3+
The generation of the adjustable inflammation of T cell, therefore originally the experimental results showed that pVAX1-TNFAIP3 has certain therapeutic effect.
The expression of Western blot detection inflammatory signals access phosphorylated protein
It is put into the homogenizer of pre-cooling, is added containing protease and inhibitors of phosphatases after mouse Colon tissue is shredded
Lysate is ground, and cracks 15min after grinding on ice, and supernatant is collected by centrifugation, and -20 DEG C save backup.According to BCA protein quantification
Method measures protein concentration, carries out 10%SDS-PAGE after albuminous degeneration and albumen is separated by electrophoresis, and electricity is gone on pvdf membrane, 3%BSA envelope
Close 1h, be added p-Erk, Erk, p-p38, p38, p-JNK, JNK, Cox-2, Cox-1, NF- κ B, p-NF- κ B, IKK β, p-IKK β,
β-actin and Foxp3 monoclonal antibody, 4 DEG C of overnight incubations, TBST are washed 3 times, each 10min；The secondary antibody of HRP label is added,
Room temperature concussion is incubated for 1h；Secondary antibody is cleaned by above-mentioned washing methods；Using Pierce ECL chemiluminescent substrate kit, with film
Development, fixing.With β-actin (1:1000) it is used as internal reference.
The GAP-associated protein GAPs such as pVAX1-TNFAIP3 intervention group and positive controls ERK, JNK, p38 and NF- κ B as the result is shown
Phosphorylation level is substantially less than model group and unloaded group (see Fig. 9 and 10), and intervention group is lower than positive controls, this shows
PVAX1-TNFAIP3 can inhibit the generation of inflammation by the activation of the signal path of inhibition related inflammation.
In addition, in summary as a result, intervention group is less compared to the expression of positive controls IL-4, IL-6, TIM1, IFN-
The expression of γ is more, and CD4+CD25+Foxp3+T cell proportion is higher, and the phosphorylation level of inflammatory signals pathway protein is also
It reduces, this imply that DNA vaccination of the invention is for existing Salicylic Acid Formulations, it is possible for the treatment of inflammatory bowel disease
Have a better effect, thus for preventing and treating inflammatory bowel disease also just with good application prospect and practical value.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Belong to those skilled in the art in the technical scope disclosed by the present invention, any changes or substitutions that can be easily thought of, all answers
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection scope in claims.
SEQUENCE LISTING
<110>Ear,nose & throat research institute of Shenzhen
<120>A kind of recombinant plasmid, DNA vaccination and its preparation method and application
<130> 9
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 2373
<212> DNA
<213> TNFAIP3
<400> 1
atggctgaac aagtccttcc tcaggctttg tatttgagca atatgcggaa agctgtgaag 60
atacgggaga gaactccaga agacattttt aaacctacta atgggatcat tcatcatttt 120
aaaaccatgc accgatacac actggaaatg ttcagaactt gccagttttg tcctcagttt 180
cgggagatca tccacaaagc cctcatcgac agaaacatcc aggccaccct ggaaagccag 240
aagaaactca actggtgtcg agaagtccgg aagcttgtgg cgctgaaaac gaacggtgac 300
ggcaattgcc tcatgcatgc cacttctcag tacatgtggg gcgttcagga cacagacttg 360
gtactgagga aggcgctgtt cagcacgctc aaggaaacag acacacgcaa ctttaaattc 420
cgctggcaac tggagtctct caaatctcag gaatttgttg aaacggggct ttgctatgat 480
actcggaact ggaatgatga atgggacaat cttatcaaaa tggcttccac agacacaccc 540
atggcccgaa gtggacttca gtacaactca ctggaagaaa tacacatatt tgtcctttgc 600
aacatcctca gaaggccaat cattgtcatt tcagacaaaa tgctaagaag tttggaatca 660
ggttccaatt tcgccccttt gaaagtgggt ggaatttact tgcctctcca ctggcctgcc 720
caggaatgct acagataccc cattgttctc ggctatgaca gccatcattt tgtacccttg 780
gtgaccctga aggacagtgg gcctgaaatc cgagctgttc cacttgttaa cagagaccgg 840
ggaagatttg aagacttaaa agttcacttt ttgacagatc ctgaaaatga gatgaaggag 900
aagctcttaa aagagtactt aatggtgata gaaatccccg tccaaggctg ggaccatggc 960
acaactcatc tcatcaatgc cgcaaagttg gatgaagcta acttaccaaa agaaatcaat 1020
ctggtagatg attactttga acttgttcag catgagtaca agaaatggca ggaaaacagc 1080
gagcagggga ggagagaggg gcacgcccag aatcccatgg aaccttccgt gccccagctt 1140
tctctcatgg atgtaaaatg tgaaacgccc aactgcccct tcttcatgtc tgtgaacacc 1200
cagcctttat gccatgagtg ctcagagagg cggcaaaaga atcaaaacaa actcccaaag 1260
ctgaactcca agccgggccc tgaggggctc cctggcatgg cgctcggggc ctctcgggga 1320
gaagcctatg agcccttggc gtggaaccct gaggagtcca ctggggggcc tcattcggcc 1380
ccaccgacag cacccagccc ttttctgttc agtgagacca ctgccatgaa gtgcaggagc 1440
cccggctgcc ccttcacact gaatgtgcag cacaacggat tttgtgaacg ttgccacaac 1500
gcccggcaac ttcacgccag ccacgcccca gaccacacaa ggcacttgga tcccgggaag 1560
tgccaagcct gcctccagga tgttaccagg acatttaatg ggatctgcag tacttgcttc 1620
aaaaggacta cagcagaggc ctcctccagc ctcagcacca gcctccctcc ttcctgtcac 1680
cagcgttcca agtcagatcc ctcgcggctc gtccggagcc cctccccgca ttcttgccac 1740
agagctggaa acgacgcccc tgctggctgc ctgtctcaag ctgcacggac tcctggggac 1800
aggacgggga cgagcaagtg cagaaaagcc ggctgcgtgt attttgggac tccagaaaac 1860
aagggctttt gcacactgtg tttcatcgag tacagagaaa acaaacattt tgctgctgcc 1920
tcagggaaag tcagtcccac agcgtccagg ttccagaaca ccattccgtg cctggggagg 1980
gaatgcggca cccttggaag caccatgttt gaaggatact gccagaagtg tttcattgaa 2040
gctcagaatc agagatttca tgaggccaaa aggacagaag agcaactgag atcgagccag 2100
cgcagagatg tgcctcgaac cacacaaagc acctcaaggc ccaagtgcgc ccgggcctcc 2160
tgcaagaaca tcctggcctg ccgcagcgag gagctctgca tggagtgtca gcatcccaac 2220
cagaggatgg gccctggggc ccaccggggt gagcctgccc ccgaagaccc ccccaagcag 2280
cgttgccggg cccccgcctg tgatcatttt ggcaatgcca agtgcaacgg ctactgcaac 2340
gaatgctttc agttcaagca gatgtatggc taa 2373
<210> 2
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 2
cggaattcca ccatggctga acaagt 26
<210> 3
<211> 25
<212> DNA
<213>It is artificial synthesized
<400> 3
ccggatatct tagccataca tctgc 25
<210> 4
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 4
cggaattcca ccatggctga acaagt 26
<210> 5
<211> 26
<212> DNA
<213>It is artificial synthesized
<400> 5
gctctagagc ttacttgtac agctcg 26
<210> 6
<211> 2373
<212> DNA
<213>Manually
<400> 6
atggctgaac aagtccttcc tcaggctttg tatttgagca atatgcggaa agctgtgaag 60
atacgggaga gaactccaga agacattttt aaacctacta atgggatcat tcatcatttt 120
aaaaccatgc accgatacac actggaaatg ttcagaactt gccagttttg tcctcagttt 180
cgggagatca tccacaaagc cctcatcgac agaaacatcc aggccaccct ggaaagccag 240
aagaaactca actggtgtcg agaagtccgg aagcttgtgg cgctgaaaac gaacggtgac 300
ggcaattgcc tcatgcatgc cacttctcag tacatgtggg gcgttcagga cacagacttg 360
gtactgagga aggcgctgtt cagcacgctc aaggaaacag acacacgcaa ctttaaattc 420
cgctggcaac tggagtctct caaatctcag gaatttgttg aaacggggct ttgctatgat 480
actcggaact ggaatgatga atgggacaat cttatcaaaa tggcttccac agacacaccc 540
atggcccgaa gtggacttca gtacaactca ctggaagaaa tacacatatt tgtcctttgc 600
aacatcctca gaaggccaat cattgtcatt tcagacaaaa tgctaagaag tttggaatca 660
ggttccaatt tcgccccttt gaaagtgggt ggaatttact tgcctctcca ctggcctgcc 720
caggaatgct acagataccc cattgttctc ggctatgaca gccatcattt tgtacccttg 780
gtgaccctga aggacagtgg gcctgaaatc cgagctgttc cacttgttaa cagagaccgg 840
ggaagatttg aagacttaaa agttcacttt ttgacagatc ctgaaaatga gatgaaggag 900
aagctcttaa aagagtactt aatggtgata gaaatccccg tccaaggctg ggaccatggc 960
acaactcatc tcatcaatgc cgcaaagttg gatgaagcta acttaccaaa agaaatcaat 1020
ctggtagatg attactttga acttgttcag catgagtaca agaaatggca ggaaaacagc 1080
gagcagggga ggagagaggg gcacgcccag aatcccatgg aaccttccgt gccccagctt 1140
tctctcatgg atgtaaaatg tgaaacgccc aactgcccct tcttcatgtc tgtgaacacc 1200
cagcctttat gccatgagtg ctcagagagg cggcaaaaga atcaaaacaa actcccaaag 1260
ctgaactcca agccgggccc tgaggggctc cctggcatgg cgctcggggc ctctcgggga 1320
gaagcctatg agcccttggc gtggaaccct gaggagtcca ctggggggcc tcattcggcc 1380
ccaccgacag cacccagccc ttttctgttc agtgagacca ctgccatgaa gtgcaggagc 1440
cccggctgcc ccttcacact gaatgtgcag cacaacggat tttgtgaacg ttgccacaac 1500
gcccggcaac ttcacgccag ccacgcccca gaccacacaa ggcacttgga tcccgggaag 1560
tgccaagcct gcctccagga tgttaccagg acatttaatg ggatctgcag tacttgcttc 1620
aaaaggacta cagcagaggc ctcctccagc ctcagcacca gcctccctcc ttcctgtcac 1680
cagcgttcca agtcagatcc ctcgcggctc gtccggagcc cctccccgca ttcttgccac 1740
agagctggaa acgacgcccc tgctggctgc ctgtctcaag ctgcacggac tcctggggac 1800
aggacgggga cgagcaagtg cagaaaagcc ggctgcgtgt attttgggac tccagaaaac 1860
aagggctttt gcacactgtg tttcatcgag tacagagaaa acaaacattt tgctgctgcc 1920
tcagggaaag tcagtcccac agcgtccagg ttccagaaca ccattccgtg cctggggagg 1980
gaatgcggca cccttggaag caccatgttt gaaggatact gccagaagtg tttcattgaa 2040
gctcagaatc agagatttca tgaggccaaa aggacagaag agcaactgag atcgagccag 2100
cgcagagatg tgcctcgaac cacacaaagc acctcaaggc ccaagtgcgc ccgggcctcc 2160
tgcaagaaca tcctggcctg ccgcagcgag gagctctgca tggagtgtca gcatcccaac 2220
cagaggatgg gccctggggc ccaccggggt gagcctgccc ccgaagaccc ccccaagcag 2280
cgttgccggg cccccgcctg tgatcatttt ggcaatgcca agtgcaacgg ctactgcaac 2340
gaatgctttc agttcaagca gatgtatggc taa 2373
<210> 7
<211> 3127
<212> DNA
<213>Manually
<400> 7
cgagctgtac aagtagatgg ctgaacaagt ccttcctcag gctttgtatt tgagcaatat 60
gcggaaagct gtgaagatac gggagagaac tccagaagac atttttaaac ctactaatgg 120
gatcattcat cattttaaaa ccatgcaccg atacacactg gaaatgttca gaacttgcca 180
gttttgtcct cagtttcggg agatcatcca caaagccctc atcgacagaa acatccaggc 240
caccctggaa agccagaaga aactcaactg gtgtcgagaa gtccggaagc ttgtggcgct 300
gaaaacgaac ggtgacggca attgcctcat gcatgccact tctcagtaca tgtggggcgt 360
tcaggacaca gacttggtac tgaggaaggc gctgttcagc acgctcaagg aaacagacac 420
acgcaacttt aaattccgct ggcaactgga gtctctcaaa tctcaggaat ttgttgaaac 480
ggggctttgc tatgatactc ggaactggaa tgatgaatgg gacaatctta tcaaaatggc 540
ttccacagac acacccatgg cccgaagtgg acttcagtac aactcactgg aagaaataca 600
catatttgtc ctttgcaaca tcctcagaag gccaatcatt gtcatttcag acaaaatgct 660
aagaagtttg gaatcaggtt ccaatttcgc ccctttgaaa gtgggtggaa tttacttgcc 720
tctccactgg cctgcccagg aatgctacag ataccccatt gttctcggct atgacagcca 780
tcattttgta cccttggtga ccctgaagga cagtgggcct gaaatccgag ctgttccact 840
tgttaacaga gaccggggaa gatttgaaga cttaaaagtt cactttttga cagatcctga 900
aaatgagatg aaggagaagc tcttaaaaga gtacttaatg gtgatagaaa tccccgtcca 960
aggctgggac catggcacaa ctcatctcat caatgccgca aagttggatg aagctaactt 1020
accaaaagaa atcaatctgg tagatgatta ctttgaactt gttcagcatg agtacaagaa 1080
atggcaggaa aacagcgagc aggggaggag agaggggcac gcccagaatc ccatggaacc 1140
ttccgtgccc cagctttctc tcatggatgt aaaatgtgaa acgcccaact gccccttctt 1200
catgtctgtg aacacccagc ctttatgcca tgagtgctca gagaggcggc aaaagaatca 1260
aaacaaactc ccaaagctga actccaagcc gggccctgag gggctccctg gcatggcgct 1320
cggggcctct cggggagaag cctatgagcc cttggcgtgg aaccctgagg agtccactgg 1380
ggggcctcat tcggccccac cgacagcacc cagccctttt ctgttcagtg agaccactgc 1440
catgaagtgc aggagccccg gctgcccctt cacactgaat gtgcagcaca acggattttg 1500
tgaacgttgc cacaacgccc ggcaacttca cgccagccac gccccagacc acacaaggca 1560
cttggatccc gggaagtgcc aagcctgcct ccaggatgtt accaggacat ttaatgggat 1620
ctgcagtact tgcttcaaaa ggactacagc agaggcctcc tccagcctca gcaccagcct 1680
ccctccttcc tgtcaccagc gttccaagtc agatccctcg cggctcgtcc ggagcccctc 1740
cccgcattct tgccacagag ctggaaacga cgcccctgct ggctgcctgt ctcaagctgc 1800
acggactcct ggggacagga cggggacgag caagtgcaga aaagccggct gcgtgtattt 1860
tgggactcca gaaaacaagg gcttttgcac actgtgtttc atcgagtaca gagaaaacaa 1920
acattttgct gctgcctcag ggaaagtcag tcccacagcg tccaggttcc agaacaccat 1980
tccgtgcctg gggagggaat gcggcaccct tggaagcacc atgtttgaag gatactgcca 2040
gaagtgtttc attgaagctc agaatcagag atttcatgag gccaaaagga cagaagagca 2100
actgagatcg agccagcgca gagatgtgcc tcgaaccaca caaagcacct caaggcccaa 2160
gtgcgcccgg gcctcctgca agaacatcct ggcctgccgc agcgaggagc tctgcatgga 2220
gtgtcagcat cccaaccaga ggatgggccc tggggcccac cggggtgagc ctgcccccga 2280
agaccccccc aagcagcgtt gccgggcccc cgcctgtgat cattttggca atgccaagtg 2340
caacggctac tgcaacgaat gctttcagtt caagcagatg tatggcggag gcggaggatc 2400
tggcggaggc ggatctatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga 2460
gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga 2520
gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa 2580
gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa 2640
ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg 2700
cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga 2760
ctcctccctg caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc 2820
ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat 2880
gtaccccgag gacggcgccc tgaagggcga gatcaagcag aggctgaagc tgaaggacgg 2940
cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc 3000
cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat 3060
cgtggaacag tacgaacgcg ccgagggccg ccactccacc ggcggcatgg acgagctgta 3120
caagtaa 3127
Claims (10)
1. a kind of recombinant plasmid, which is characterized in that including TNFAIP3 gene and eukaryotic expression vector pVAX1, the TNFAIP3
The GenBank accession number of gene is KJ892292.1.
2. the preparation method of recombinant plasmid described in claim 1, which is characterized in that specific step is as follows：It will include TNFAIP3
The segment of gene is inserted between EcoR I and EcoR the V restriction enzyme site of carrier pVAX1.
3. application of the recombinant plasmid described in claim 1 in the drug that preparation prevents and treats inflammatory bowel disease.
4. a kind of DNA vaccination, which is characterized in that including recombinant plasmid described in claim 1.
5. DNA vaccination according to claim 4, which is characterized in that the vaccine is multi-phase emulsion.
6. DNA vaccination according to claim 4 or 5, which is characterized in that further include poly lactide-glycolide acid, poly-
Vinyl alcohol.
7. a kind of preparation method of DNA vaccination, which is characterized in that include the following steps：
It takes poly lactide-glycolide acid to be dissolved in organic solvent, forms organic phase；
The solution for taking recombinant plasmid described in claim 1 is mixed to form lotion with the organic phase；
The lotion is mixed with polyvinyl alcohol water solution, forms multi-phase emulsion.
8. preparation method according to claim 7, which is characterized in that further include：Deionization is added in the multi-phase emulsion
Water, after organic solvent volatilization, precipitating is collected in centrifugation.
9. DNA vaccination made from claim any one of 7-8.
10. claim 4-6,9 described in any item DNA vaccinations answering in the drug that preparation prevents and treats inflammatory bowel disease
With.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988779A (en) * | 2019-01-30 | 2019-07-09 | 刘志强 | Recombinant plasmid, DNA vaccination and its preparation method and application |
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