CN109988779A - Recombinant plasmid, DNA vaccination and its preparation method and application - Google Patents
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Abstract
The invention discloses a kind of recombinant plasmid, corresponding DNA vaccination and preparation method and its applications in the drug of preparation prevention or treatment anaphylactia.The recombinant plasmid includes Der p2 gene and A20 gene, and the DNA vaccination can prepare with the following method then by including that the raw material of the recombinant plasmid is made: poly lactide-glycolide acid being taken to be dissolved in organic solvent, form organic phase；The solution for taking recombinant plasmid is mixed to form colostrum with organic phase；Colostrum is mixed with polyvinyl alcohol water solution, forms multi-phase emulsion.Recombinant plasmid and DNA vaccination provided by the present invention are shown experimentally that it can be such that Th2 immune response converts to the direction Th1.Meanwhile this recombinant plasmid and DNA vaccination can inhibit the generation of inflammation by up-regulation regulatory T cells.
Description
Technical field
The present invention relates to biological immunology field, more particularly, to a kind of recombinant plasmid, DNA vaccination and preparation method thereof and
Using.
Background technique
Anaphylactia is common disease clinically, specifically includes allergic rhinitis, allergic asthma etc..Wherein anaphylaxis
Rhinitis (Allergic rhinitis, AR) is a kind of respiratory tract local immunity inflammatory reaction, and morbidity is mainly characterized by Th2
Allergen specific immune response based on type cell, while generating specific IgE antibody.According to epidemiological survey, dust mite
It is the important allergen for causing China AR, there are about the upper respiratory tract autopaths of 70-80% to be positive to dust mite, wherein
Der p2 is 87.8% to upper respiratory tract autopath's immunodiagnosis positive rate, is most important dust mite allergen.Allergia nose
Scorching disease incidence is higher and shows the trend risen year by year, but difficult in a manner of the current mainly symptomatic treatment based on antihistamine
To obtain satisfied curative effect, must from the point of view of still lack effective treatment means.
In recent years, the rapid development of DNA vaccination technology represents new Vaccine Development approach and research direction.DNA vaccination
Principle be that the plasmid of foreign gene-carrying is directly injected into human or animal's body, express that it in vivo, activate siberian crabapple
System, the immune response of inducing specific.DNA vaccination has the advantages that without extracting target protein, in vitro protokaryon or eukaryon table
It reaches, expression product is purified and is processed；Easy to operate and thermal stability；To there is the time in the foreign gene of carrying long in vivo,
The time for expressing foreign protein is long, can continue stimulation immune system；Vaccine delivery method can be used using a variety of.
Although at present to including allergic rhinitis anaphylactia and gene therapy and DNA vaccination etc. have it is more
Disclosure, still, the DNA vaccination in relation to allergic rhinitis, especially can pass through induction immune cell responses strategy use
Gene therapy vaccine carrys out the problems such as directly effectively being treated, and there are no corresponding reports in the prior art.Therefore it provides
A kind of DNA vaccination that can treat allergic rhinitis is still related researcher's urgent problem.
Summary of the invention
A technical problem to be solved by this invention is how to provide a kind of recombinant plasmid and its in preparation prevention or control
It treats application, corresponding DNA vaccination and preparation method in the drug of anaphylactia and its prevents or treat anaphylaxis in preparation
Application in the drug of disease.
The technical scheme adopted by the invention is that:
According to the first aspect of the invention, the invention proposes a kind of recombinant plasmids, including Der p2 gene and A20 base
Cause, wherein the GenBank accession number of Der p2 gene is FM177223.1, and the GenBank accession number of the A20 gene is
KJ892292.1.Wherein, A20 be it is a kind of with the ubiquitin of ubiquitination and deubiquitination double enzyme active function modify zymoprotein,
It plays an important role in adjusting immunity of organism and inflammatory reaction.
Preferably, the carrier of recombinant plasmid is carrier for expression of eukaryon.
It is further preferred that carrier for expression of eukaryon is pVAX1, pVAX1 is a kind of common mammalian expression vector, is permitted
Perhaps transient expression albumen at a high level in mammalian cells.In some embodiments of the invention, can by Der p2 and
The gene of A20 is that restriction enzyme site is inserted into expression vector pVAX1 with Eco R I and Not I, obtains recombinant plasmid pVAX1-Der
p2-A20。
According to the second aspect of the invention, the invention also provides a kind of DNA vaccination, the recombination including any of the above-described
Plasmid.
It preferably, further include pharmaceutically acceptable immunologic adjuvant.
It is further preferred that pharmaceutically acceptable immunologic adjuvant is poly lactide-glycolide acid (PLGA).
Preferably, DNA vaccination is at least one of multi-phase emulsion or microballoon.
In some embodiments of the invention, DNA vaccination can be used for real in a manner of injection, mucous membrane, particle gun importing etc.
It applies immune；Specifically, can be used for include but are not limited to the side such as intravenous injection, intra arterial injection, intramuscular injection, subcutaneous injection
Formula is implemented immune.Wherein, the DNA vaccination of multi-phase emulsion can direct intranasal administration, more directly act rapidly on schneiderian membrance office
Portion induces part and systemic immunity response.
According to the third aspect of the present invention, the invention also provides the preparation methods of above-mentioned DNA vaccination, including following step
It is rapid:
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 colostrum with organic phase；
Colostrum is mixed with polyvinyl alcohol water solution, forms multi-phase emulsion.
The W1/O/W2 type multi-phase emulsion as made from this method can direct intranasal administration, directly act on nose, be not necessarily to
Intramuscular injection more directly can promptly have an effect.
Preferably, further include the organic solvent removed in multi-phase emulsion, form microballoon.
According to the fourth aspect of the present invention, it is pre- in preparation that the invention also provides above-mentioned recombinant plasmid or DNA vaccinations
Application in anti-or treatment anaphylactia drug.
Preferably, anaphylactia is allergic rhinitis.
The beneficial effects of the present invention are:
Recombinant expression and corresponding DNA vaccination while the present invention creatively constructs Der p2 and A20, in reality
Der p2 specific antibody, cell factor IL-4/IL-13/TNF- alpha levels, schneiderian membrane Histopathology change in the experiment on border
Significantly reduce, IgG1, IgG2a, IL-10, IFN-γ, TGF-β 1 it is horizontal it is significant increase, show that recombinant plasmid and DNA vaccination can be with
Convert Th2 immune response to the direction Th1.CD4+CD25+Foxp3+T cell proliferation is obvious, shows of the invention
Recombinant plasmid and DNA vaccination can inhibit the generation of inflammation by up-regulation regulatory T cells.These all confirm that the present invention is mentioned
The recombinant plasmid and DNA vaccination of confession have good therapeutic effect to anaphylactia.
Detailed description of the invention
Fig. 1 is the flow chart of the building of AR model mice and administration time in one embodiment of the present of invention.
Fig. 2 is the external plasmid construction of pVAX1-Der p2-A20 (pDA) and nano-packaging in the embodiment of Fig. 1 of the invention
Result figure.Wherein, 2A is double digestion as a result, 2B is sequencing result, and 2C is the Electronic Speculum of nano-packaging as a result, 2D (I) and 2D (II)
For the result of in-vitro transfection 293T cell.
Fig. 3 is that the expression, purifying of dermatophagoides pteronyssinus recombinant allergen Der p2 and immunology are living in the embodiment of Fig. 1 of the invention
Property qualification result.Wherein, 3A is to recombinate the identification of Der p2 expression plasmid as a result, 3B is the inducing expression for recombinating Der p2 albumen
As a result (M: albumen marker；1: before induction；2: after induction；3: supernatant；4: precipitating), 3C is the affine layer for recombinating Der p2 albumen
Analysis is as a result, 3D is the SDS-PAGE result (M: albumen Marker of recombinant protein Der p2 when eluting peak；1: when eluting initial；
2: when elution peak), 3E is the immunogenicity (NC: Healthy Human Serum of ELISA detection Der p2 albumen；AR: dermatophagoides pteronyssinus allergy is suffered from
Person's positive serum), 3F is the allergenicity (M: albumen Marker of Western blot detection Der p2 albumen；1: Healthy People blood
Clearly；2: dermatophagoides pteronyssinus autopath positive serum).
Fig. 4 be NC group in the embodiment of Fig. 1 of the invention, AR group, PpDA group, PLGA group, pDA group, pD group mouse row
Change appraisal result to learn.Wherein, 4A is that the nose of grabbing of each group mouse scores, and 4B is the sneezing scoring of each group mouse.
Fig. 5 is each group mouse schneiderian membrane HE colored graph (200 ×) in Fig. 1 embodiment of the invention, and 5A-5F respectively represents NC
The coloration result of group, AR group, PpDA group, PLGA group, pDA group, pD group.
Fig. 6 is the testing result figure of Der p2 specific antibody in each group mice serum in the embodiment of Fig. 1 of the invention,
6A-6C respectively indicates IgE antibody, IgG1 antibody, IgG2a antibody.
Fig. 7 is the expression water of cell factor in each group mouse spleen lymphocyte supernatant in the embodiment of Fig. 1 of the invention
Flat result figure, 7A-7F respectively indicate the expression of IFN-γ, IL-4, IL-10, IL-13, TGF-β 1, TNF-α.
Fig. 8 is each group mice spleen monocyte CD4 in the embodiment of Fig. 1 of the invention+CD25+Foxp3+The streaming of T cell
Result figure.
Specific embodiment
It is carried out below with reference to technical effect of the embodiment to design of the invention, specific structure and generation clear, complete
Ground description, to be completely understood by the purpose of the present invention, feature and effect.Obviously, described embodiment is of the invention one
Section Example, rather than whole embodiments, based on the embodiment of the present invention, those skilled in the art are not paying creativeness
Other embodiments obtained, belong to the scope of protection of the invention under the premise of labour.
1. experimental method
Female is selected, 6-8 week old, is purchased from Guangdong Medical Lab Animal Center by Balb/c healthy mice 60.Random point
At 6 groups, every group 10, it is divided into control group (NC), model group (AR), nano vaccine treatment group (PpDA), blank nanometer treatment group
(PLGA), 1 treatment group of gymnoplasm grain (pDA) and 2 treatment group of gymnoplasm grain (pD), the processing mode of every group of experimental animal are as shown in table 1.
Every group of wherein 5 mouse take its complete nasal cavity as HE dye, every group remaining 5 take its schneiderian membrane for protein expression surveys
It is fixed.
The grouping of 1 experimental animal of table and disposition
Target gene Der p2 and A20 are linked together by linker, inserted with the clonal fashion of Eco RI and Not I
Enter into purpose carrier pVAX1, finally by screening, extracts plasmid and sequencing company is sent to be sequenced, verify target recombinant plasmid
The accuracy of (pVAX1-Der p2-A20 carrier for expression of eukaryon, pDA).Another recombinant plasmid is obtained in the same way
(pVAX1-Der p2 carrier for expression of eukaryon, pD).
Primer is according to (the GenBank accession number of Der p2 gene is FM177223.1, the GenBank accession number of A20 gene
For the design of KJ892292.1) gene coded sequence, the synthesis of commission Shanghai Sheng Gong bioengineering Co., Ltd, specific primer information is shown in
Under:
Der p2 amplimer:
Upstream primer (SEQ ID No.1): AGCCGTTTTCGAAGCCAACC；
Downstream primer (SEQ ID No.2): GGATCGATACCGGGAACATCAAC.
A20 amplimer:
Upstream primer (SEQ ID No.3): CGGAATTCCACCATGGCTGAACAAGT；
Downstream primer (SEQ ID No.4): CCGGATATCTTAGCCATACATCTGC.
Using supersound method, it is to contain material with poly lactide-glycolide acid (PLGA), prepares PLGA-
PVAX1-Der p2-A20 (PpDA) DNA vaccination.It weighs 100mg PLGA to be dissolved in 900 μ L methylene chloride and 100 μ L acetone, shape
At organic phase；100 μ L are taken to be dissolved in the pVAX1-Der p2-A20 gymnoplasm grain 1 (concentration is 10 μ g/ μ L) of sterile ultrapure water, as interior
Water phase；By organic phase and inner aqueous phase two-phase mixtures, in ice-water bath, ultrasonication 25 (power 40W, intermittent times/continuous
Working time is 6s/6s), form milky colostrum；2% polyvinyl alcohol (PVA) aqueous solution of 2mL is slowly added dropwise in colostrum
As outer aqueous phase, ultrasound is carried out with identical condition again, forms multiphase emulsion.50mL deionization is added in multiphase emulsion
Water is stirred at room temperature (or rotary evaporation) 4h and volatilizees completely to organic solvent；4 DEG C, 10000rpm, it is centrifuged 20min, collects precipitating,
Sterile washing 3 times；Freeze-drying, -80 DEG C save backup.
With the BL21 engineering of a large amount of inducing expression plasmids of p2 containing pET28a-Der of isopropylthiogalactoside (IPTG)
Bacterium, expression product exist in the form of recombinant protein inclusion body, wash through inclusion body with after dissolution, pass through Ni2+Affinity chromatography
Recombinant protein Der p2 is purified, after ultrafiltration membrance filter, displacement, with sodium dodecyl sulphate-polyacrylamide gel electricity
Swimming (SDS-PAGE) purification Identification product.Der is analyzed through ELISA and Western blot with dermatophagoides pteronyssinus autopath positive serum
The immunological characteristic of p2 recombinant protein.
Fig. 1 is the flow chart of the building and administration time of AR model mice of the invention.As shown in Figure 1, the system of AR model
Standby process is specific as follows:
Respectively in 0,7,14 day sensitized mice, AR group, PpDA group, PLGA group, pDA group, pD group are every time through intraperitoneal injection 100
μ g Der p2 recombinant protein and 15 μ g, NC group of cholera toxin (CT) are replaced with PBS.After sensitization terminates 1 week, PpDA group, PLGA
Group, pDA group, pD group respectively with PpDA nano vaccine (contain 100 μ g pVAX1-Der p2-A20 plasmids), PLGA nanometers of blank it is micro-
Grain, 100 μ g pVAX1-Der p2-A20 plasmids and 100 μ g pVAX1-Der p2 plasmids carried out Nasal immunization treatment, every 3 days
It collunarium 1 time, treats 5 times altogether.With Der p2 recombinant protein (200 μ g), collunarium excites mouse respectively after last therapeutic, 1 time a day,
For three days on end, excitation terminates to put to death mouse afterwards for 24 hours.PpDA nano vaccine used in scheme is " 1.3DNA vaccine PLGA-
The nano particle that DNA vaccination made from the preparation of pVAX1-Der p2-A20 (PpDA) " is dissolved with PBS, can directly collunarium give
Medicine directly acts on allergic rhinitis pathogenic site, is not necessarily to intramuscular injection.
2. experimental result
Der p2 and the A20 full-length gene amplified carries out sequence alignment with 1.0 software of DNAssist after being sequenced, knot
Fruit shows Der p2 and the A20 complete encoding sequence complete one announced in the Der p2 gene obtained and A20 gene and Genbank
It causes.Fig. 2 is target construction of recombinant plasmid and nano-packaging result figure.Wherein, 2A is double digestion as a result, 2B is sequencing result.Such as
Shown in 2A and 2B, band and target gene fragment size that target recombinant plasmid pVAX1-Der p2-A20 is generated after double digestion
Unanimously；The positive colony bacterium identified shows Der p2 base in the recombination pVAX1-Der p2-A20 of acquisition after being sequenced
Sequence in cause and A20 gene and Genbank carries out sequence analysis, and the sequence of clone and the full length sequence homology of announcement are
100%, show that eukaryon expression plasmid pVAX1-Der p2-A20 is constructed successfully.
Fig. 2 is target construction of recombinant plasmid and nano-packaging result figure, wherein 2C is the Electronic Speculum of nano-packaging as a result, 2D
(I) and 2D (II) be in-vitro transfection 293T cell result.As shown in 2C, PpDA nano DNA vaccine is under scanning electron microscope in ellipse
Round, smooth particle, average grain diameter 300-500nm, uniform in size, favorable dispersibility.It is observed under Laser Scanning Confocal Microscope
Shown in uptake ratio such as 2D (I) and 2D (II) of the 293T cell to nano vaccine.
Fig. 3 is the expression, purifying and immunologic competence qualification result of dermatophagoides pteronyssinus recombinant allergen Der p2.Wherein, 3A is
Recombinate that Der p2 expression plasmid is identified as a result, 3B is the inducing expression result (M: albumen marker for recombinating Der p2 albumen；1:
Before induction；2: after induction；3: supernatant；4: precipitating), 3C is to recombinate the affinity chromatography of Der p2 albumen as a result, 3D is elution peak
When recombinant protein Der p2 SDS-PAGE result (M: albumen Marker；1: when eluting initial；2: when elution peak), 3E is
Immunogenicity (the NC: Healthy Human Serum of ELISA detection Der p2 albumen；AR: dermatophagoides pteronyssinus autopath positive serum), 3F is
The allergenicity (M: albumen Marker of Western blot detection Der p2 albumen；1: Healthy Human Serum；2: dermatophagoides pteronyssinus allergy is suffered from
Person's positive serum).As shown in figure 3, this Success in Experiment constructs prokaryotic expression plasmid pET28a (+)-Der p2, which is turned
Change E.coli BL21 inducing expression, after affinitive layer purification, SDS-PAGE display obtains destination protein, ELISA and Western
Blot verifies it can be in conjunction with the specific IgE of dermatophagoides pteronyssinus autopath's positive serum, it was demonstrated that the Der of inducing expression after purification
P2 recombinant protein has immunologic competence.
After the excitation of last nasal cavity, the behaviouristics observed in mouse 30min changes, and record grabs the number of nose and sneezing.Note
Minute mark standard is respectively as follows:
Grab nose: grabbing nose incessantly is 3 points, and frequently grabbing nose (10-20 times/min) is 2 points, and slightly grabbing nose is 1 point, is 0 without nose is grabbed
Point；
Sneeze: 11 are sneezed the above are 3 points, 4~10 are 2 points, and 1~3 is 1 point, and not making is 0 score.
Total score is recorded using the addition method, total score > 5, which divides, indicates modeling success.
Fig. 4 is NC group, AR group, PpDA group, PLGA group, pDA group, the behaviouristics of pD group mouse change appraisal result.Wherein,
4A is that the nose of grabbing of each group mouse scores, and 4B is the sneezing scoring of each group mouse.As shown in figure 4, NC group occasionally grabs nose, AR group is grabbed
Nose and sneezing symptom are obvious, and the equal > 5 of score divides, and show modeling success.
Compared with AR group, the allergic symptom of PpDA group, pDA group and pD group is substantially reduced, wherein being mitigated with PpDA group most bright
Aobvious, PLGA group nothing is substantially change；The symptom score of PpDA group, pDA group and pD group is significantly lower than AR group, and wherein PpDA group symptom is commented
Divide to reduce and become apparent from, PLGA group is without substantially changeing compared with AR group, and two kinds of scorings of pDA group are all than two kinds of scorings of pD group
It is low, especially sneezing scoring.This shows that PpDA nano vaccine can be obviously improved the allergic symptom of allergic rhinitis mouse, and
Expression becomes apparent the improvement of allergic symptom compared to individual Der p2 while Der p2 and A20.
Mouse excites in last and is anaesthetized disconnected neck execution afterwards for 24 hours, takes out the complete nasal cavity of mouse (n=5), is fixed on 4%
In paraformaldehyde for 24 hours, it is de- that suitable vinegar hydrochloric acid formaldehyde decalcifying Fluid, decalcification 6h, then the ethanol gradient through various concentration are prepared later
Water, paraffin embedding, conventional section, with a thickness of 5um, using Hematoxylin-eosin dyeing (HE dyeing), light under the microscope, is taken pictures afterwards.
Fig. 5 is each group mouse schneiderian membrane HE colored graph (200 ×), and 5A-5F respectively represents NC group, AR group, PpDA group, PLGA group, pDA
The coloration result of group, pD group.As shown in figure 5, AR group and PLGA group mouse schneiderian membrance oedema, blood vessel dilatation, glandular hyperplasia, inherently
Visible a large amount of eosinophils in layer；NC group mouse schneiderian membrance has no apparent inflammatory reaction；PpDA group, pDA group and
The schneiderian membrance inflammatory reaction of pD group significantly mitigates compared with AR group, wherein it is most obvious with the mitigation of PpDA group, and the degree of alleviation of pDA group
It is more obvious than pD group.The above results show that expression is received compared to individual PpD while Der p2 and A20 in PpDA nano vaccine
Rice vaccine might have more obvious therapeutic effect for allergic rhinitis mouse.
Blood about 1mL is taken through eyeball of mouse rear vein beard, indirect elisa method detects Der p2 allergen specificity in serum
IgE, IgG1 and IgG2a.Der p2 allergy primordial covering is recombinated with 5 μ g/mL, 4 DEG C overnight, 3%BSA/PBS closing.Use 1%BSA/
PBST dilutes mice serum (IgE, 1:5, IgG1,1:2000 and IgG2a, 1:1000), 37 DEG C of incubation 1h.After board-washing, add
Secondary antibody (IgE, IgG1 and IgG2a), 37 DEG C of incubation 1h.Board-washing again is added tmb substrate and is protected from light colour developing 10-15min.Terminate bottom
Object reaction after and immediately at 450nm reading OD value.
Fig. 6 is the result figure of Der p2 specific antibody in each group mice serum in the embodiment of Fig. 1 of the invention, 6A-
6C respectively indicates IgE antibody, IgG1 antibody, IgG2a antibody.As shown in fig. 6, compared with NC group, in AR group and PLGA group serum
Der p2 specific IgE (Der p2-sIgE) level obviously increases, and illustrates modeling success.Compared with AR group, PpDA group, pDA
Der p2-sIgE is substantially reduced in the serum of group and pD group, wherein become apparent from the reduction of PpDA group, and pDA group is compared to pD
Group reduces ground clearly.Compared with AR group, PpDA group is significantly increased with IgG1 and IgG2a level in pDA group serum, wherein
It is become apparent from the raising of PpDA group, and pDA group also has compared to pD group and obviously increases；In PLGA group serum IgG1 and
IgG2a level is without significant difference compared with AR group, and the raising of PpDA group becomes apparent from, and pDA group also has clearly compared to pD group
Raising.The testing result of these three comprehensive allergen specificity antibodies is it can be shown that PpDA nano vaccine has certain control
Therapeutic effect, and expressing while Der p2 and A20 compared to individual Der p2 is highly significant for the promotion of therapeutic effect
's.
Separating mouse spleen lymphocyte under aseptic condition cultivates 72h with RPMI1640 culture medium in constant incubator,
Supernatant is collected by centrifugation.Using the water of IFN-γ, IL-4, IL-10, IL-13, TGF-β 1 and TNF-α in ELISA measurement supernatant
Flat, operating procedure is carried out in strict accordance with kit specification.
Fig. 7 is the expression result figure of cell factor in each group mouse spleen lymphocyte supernatant, and 7A-7F is respectively indicated
IFN-γ, IL-4, IL-10, IL-13, TGF-β 1, TNF-α expression.As shown in fig. 7, AR group spleen is thin compared with NC group
IL-4 and IL-13 is horizontal in born of the same parents' culture supernatant significantly increases, and illustrates sensitization success.Compared with AR group, PpDA group, pDA group and pD
IL-4, IL-13 and TNF-α level significantly reduce in the spleen cell cultures supernatant of group, and IFN-γ, IL-10 and TGF-β 1 are horizontal aobvious
Work increases, and becomes apparent from wherein being increased with PpDA group, and PLGA group compared with AR group without significant difference.Show PpDA nano vaccine energy
The inflammatory reaction for reducing allergic rhinitis mouse can be such that Th2 immune response converts to the direction Th1.
Cell concentration is adjusted to 1 × 10 by separating mouse spleen lymphocyte under aseptic condition6/mL.First use APC Rat
Anti-Mouse CD4 and FITC Rat Anti-Mouse CD25 streaming antibody dyes 15min, is then fixed with paraformaldehyde
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.As a result as shown in Figure 8.Compared with AR group, PpDA group and pDA group CD4+CD25+Foxp3+T cell ratio increases, wherein with
PpDA group increase becomes apparent from, and PLGA group compared with AR group without significant difference.Due to CD4+CD25+Foxp3+T cell is adjustable
The generation of inflammation, therefore originally the experimental results showed that PpDA nano vaccine has certain therapeutic effect.
In summary as a result, compared with pDA group, level lower, IL-10, IFN- of PpDA group IL-4, IL-13, TNF-α
More, the CD4 of level of γ, TGF-β 1+CD25+Foxp3+T cell ratio is higher, this imply that of the invention after PLGA is encapsulated
PVAX1-Der p2-A20 nano DNA vaccine treated compared to gymnoplasm grain (pVAX1-Der p2-A20) directly Nasal immunization
Allergic rhinitis effect is more preferable, thus for prevent and treat allergic rhinitis also just with good application prospect with practical valence
Value.And by pDA group compared with pD group, comprehensive behaviouristics changes, histopathology and allergen specificity antibody and other
Expression generates the effect mutually cooperateed with while some comparison results can be seen that Der p2 and A20 again, effectively adjusts and is immunized
Response has the remission effect of illness compared to individual Der p2 and is obviously promoted, to can make Der p2-
A20 nano DNA vaccine is compared to individual Der p2 nano DNA vaccine for treating anaphylactia, especially allergia nose
Inflammation also has therapeutic effect more outstanding.
It is to be illustrated to presently preferred embodiments of the present invention, but the present invention is not limited to the embodiment above,
Those skilled in the art can also make various equivalent deformation or replacement on the premise of without prejudice to spirit of the invention, this
Equivalent deformation or replacement are all included in the scope defined by the claims of the present application a bit.
SEQUENCE LISTING
<110>ear,nose & throat research institute, Liu Zhiqiang Shenzhen
<120>recombinant plasmid, DNA vaccination and its preparation method and application
<130> 9
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
agccgttttc gaagccaacc 20
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<400> 2
ggatcgatac cgggaacatc aac 23
<210> 3
<211> 26
<212> DNA
<213>artificial sequence
<400> 3
cggaattcca ccatggctga acaagt 26
<210> 4
<211> 25
<212> DNA
<213>artificial sequence
<400> 4
ccggatatct tagccataca tctgc 25
Claims (10)
1. a kind of recombinant plasmid, which is characterized in that including Der p2 gene and A20 gene, the GenBank of the Der p2 gene
Accession number is FM177223.1, and the GenBank accession number of the A20 gene is KJ892292.1.
2. recombinant plasmid according to claim 1, which is characterized in that the carrier of the recombinant plasmid is eukaryotic expression load
Body, preferably pVAX1.
3. a kind of DNA vaccination, which is characterized in that the DNA vaccination includes the described in any item recombinant plasmids of claim 1-2.
4. DNA vaccination according to claim 3, which is characterized in that further include pharmaceutically acceptable immunologic adjuvant.
5. DNA vaccination according to claim 4, which is characterized in that the pharmaceutically acceptable immunologic adjuvant is poly- cream
Acid-co-glycolic acid.
6. according to the described in any item DNA vaccinations of claim 3-5, which is characterized in that the DNA vaccination is multi-phase emulsion, micro-
At least one of ball.
7. the preparation method of DNA vaccination described in claim 5, which comprises the following steps:
It takes poly lactide-glycolide acid to be dissolved in organic solvent, forms organic phase；
The solution for taking the described in any item recombinant plasmids of claim 1-2 is mixed to form colostrum with the organic phase；
The colostrum is mixed with polyvinyl alcohol water solution, forms multi-phase emulsion.
8. the preparation method of DNA vaccination according to claim 7, which is characterized in that further include removing the multi-phase emulsion
In the organic solvent, formed microballoon.
9. the described in any item recombinant plasmids of claim 1-2 or the described in any item DNA vaccinations of claim 3-6 are pre- in preparation
Application in anti-or treatment anaphylactia drug.
10. application according to claim 9, which is characterized in that the anaphylactia is allergic rhinitis.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5958891A (en) * | 1996-04-24 | 1999-09-28 | Hsu; Ching-Hsiang | Recombinant eukaryotic plasmids containing allergen-gene and use thereof for the prevention and/or treatment of allergic diseases |
| CN1705492A (en) * | 2002-08-29 | 2005-12-07 | 新加坡国立大学 | Recombinant nucleic acid useful for inducing protective immune response against allergens |
| CN106282213A (en) * | 2015-06-03 | 2017-01-04 | 深圳北京大学香港科技大学医学中心 | A kind of method expressing also purification of soluble dermatophagoides pteronyssinus main allergen Der p 2 albumen |
| CN108893488A (en) * | 2018-06-19 | 2018-11-27 | 刘志强 | A kind of recombinant plasmid, DNA vaccination and its preparation method and application |
-
2019
- 2019-01-30 CN CN201910091253.7A patent/CN109988779B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5958891A (en) * | 1996-04-24 | 1999-09-28 | Hsu; Ching-Hsiang | Recombinant eukaryotic plasmids containing allergen-gene and use thereof for the prevention and/or treatment of allergic diseases |
| CN1705492A (en) * | 2002-08-29 | 2005-12-07 | 新加坡国立大学 | Recombinant nucleic acid useful for inducing protective immune response against allergens |
| CN106282213A (en) * | 2015-06-03 | 2017-01-04 | 深圳北京大学香港科技大学医学中心 | A kind of method expressing also purification of soluble dermatophagoides pteronyssinus main allergen Der p 2 albumen |
| CN108893488A (en) * | 2018-06-19 | 2018-11-27 | 刘志强 | A kind of recombinant plasmid, DNA vaccination and its preparation method and application |
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| 胡文会等: "锌指蛋白A20在变应性鼻炎小鼠模型鼻黏膜中的表达", 《广东医学》 * |
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