CN109988779B - Recombinant plasmid, DNA vaccine, preparation method and application thereof - Google Patents
Recombinant plasmid, DNA vaccine, preparation method and application thereof Download PDFInfo
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- CN109988779B CN109988779B CN201910091253.7A CN201910091253A CN109988779B CN 109988779 B CN109988779 B CN 109988779B CN 201910091253 A CN201910091253 A CN 201910091253A CN 109988779 B CN109988779 B CN 109988779B
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a recombinant plasmid, a corresponding DNA vaccine, a preparation method and application thereof in preparing medicines for preventing or treating allergic diseases. The recombinant plasmid comprises Der p2 gene and A20 gene, and the DNA vaccine is prepared from the raw materials comprising the recombinant plasmid, and can be prepared by the following method: dissolving polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase; mixing the solution of the recombinant plasmid with an organic phase to form colostrum; mixing the colostrum with polyvinyl alcohol water solution to form multiphase emulsion. Experiments show that the recombinant plasmid and the DNA vaccine provided by the invention can convert Th2 immune response to Th 1. Meanwhile, such recombinant plasmids and DNA vaccines can suppress the occurrence of inflammation by up-regulating regulatory T cells.
Description
Technical Field
The invention relates to the field of biological immunology, in particular to a recombinant plasmid, a DNA vaccine, a preparation method and application thereof.
Background
Allergic diseases are common clinical diseases, and specifically include allergic rhinitis, allergic asthma, and the like. Among them, allergic rhinitis (Allergic rhinitis, AR) is a local immune inflammatory reaction of the respiratory tract, and its onset is mainly characterized by allergen-specific immune reaction of Th2 type cells as well as production of specific IgE antibodies. According to epidemiological investigation, the dust mites are important allergens for causing AR in China, about 70-80% of patients with upper respiratory tract allergy react positively to the dust mites, wherein the immunodiagnosis positive rate of Der p2 to the patients with upper respiratory tract allergy is 87.8%, and the dust mites are the most main dust mite allergens. The incidence of allergic rhinitis is high and the trend of rising year by year, but the satisfactory curative effect is difficult to obtain in the current symptomatic treatment mode mainly comprising antihistamine, and the effective treatment means still cannot be seen in general.
In recent years, the rapid development of DNA vaccine technology represents a new approach and direction of vaccine development. The principle of DNA vaccine is that plasmid carrying exogenous gene is directly injected into human body or animal body to make it express in living body, activate immune system and induce specific immune response. The DNA vaccine has the following advantages: the target protein is not required to be extracted, prokaryotic or eukaryotic expression is performed in vitro, and the expression product is purified and processed; easy operation and thermal stability; the carried exogenous gene has long in vivo existence time, long expression time of exogenous protein and can continuously stimulate the immune system; a variety of vaccine delivery methods can be employed.
Although there are many disclosures of allergic diseases including allergic rhinitis, gene therapy, DNA vaccines, etc., there are no corresponding reports in the prior art on the problems of DNA vaccines for allergic rhinitis, especially whether effective treatment can be directly performed by using gene therapeutic vaccines through a strategy of inducing immune cell responses. Thus, the provision of a DNA vaccine capable of treating allergic rhinitis remains a problem that has been urgently addressed by related researchers.
Disclosure of Invention
The invention aims to solve the technical problem of providing a recombinant plasmid and application thereof in preparing medicaments for preventing or treating allergic diseases, a corresponding DNA vaccine and a preparation method thereof, and application thereof in preparing medicaments for preventing or treating allergic diseases.
The technical scheme adopted by the invention is as follows:
according to a first aspect of the present invention, the present invention provides a recombinant plasmid comprising a Der p2 gene and an a20 gene, wherein the Der p2 gene has GenBank accession No. FM177223.1, and the a20 gene has GenBank accession No. KJ892292.1. Wherein A20 is ubiquitin-modified enzyme protein with ubiquitin and deubiquitination double enzyme activities, and plays an important role in regulating organism immunity and inflammatory reaction.
Preferably, the vector of the recombinant plasmid is a eukaryotic expression vector.
Further preferred, the eukaryotic expression vector is pVAX1, pVAX1 being a commonly used mammalian expression vector allowing for high levels of transient expression of the protein in mammalian cells. In some embodiments of the invention, the genes of Der p2 and A20 can be inserted into the expression vector pVAX1 with Eco R I and Not I as cleavage sites to obtain the recombinant plasmid pVAX1-Der p2-A20.
According to a second aspect of the invention, the invention also provides a DNA vaccine comprising a recombinant plasmid according to any one of the above.
Preferably, a pharmaceutically acceptable immunological adjuvant is also included.
Further preferably, the pharmaceutically acceptable immunological adjuvant is polylactic acid-glycolic acid copolymer (PLGA).
Preferably, the DNA vaccine is at least one of a heterogeneous emulsion or microsphere.
In some embodiments of the invention, DNA vaccines may be used to administer immunity by injection, mucosal, gene gun introduction, and the like; in particular, it can be used to administer immunity in ways that include, but are not limited to, intravenous, arterial, intramuscular, subcutaneous, and the like. Wherein, the DNA vaccine of the multiphase emulsion can be directly and nasally administrated, and can be more directly and rapidly acted on the local part of nasal mucosa to induce local and systemic immune response.
According to a third aspect of the present invention, the present invention also provides a method for preparing the DNA vaccine described above, comprising the steps of:
dissolving polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase;
mixing the solution of the recombinant plasmid with an organic phase to form colostrum;
mixing the colostrum with polyvinyl alcohol water solution to form multiphase emulsion.
The W1/O/W2 type multiphase emulsion prepared by the method can be directly used for nasal administration by dripping, directly acts on the nose, does not need intramuscular injection, and can act more directly and rapidly.
Preferably, the method further comprises removing the organic solvent in the multiphase emulsion to form microspheres.
According to a fourth aspect of the present invention, the present invention also provides the use of the recombinant plasmid or DNA vaccine described above in the preparation of a medicament for the prevention or treatment of allergic diseases.
Preferably, the allergic disease is allergic rhinitis.
The beneficial effects of the invention are as follows:
the invention creatively constructs the simultaneous expression recombinant plasmid of Der p2 and A20 and the corresponding DNA vaccine, and in practical experiments, the Der p2 specific antibody, the cytokine IL-4/IL-13/TNF-alpha level and the pathological histology change of nasal mucosa are obviously reduced, igG1, igG2a and IL-10. IFN-gamma and TGF-beta 1 levels are significantly elevated, indicating that recombinant plasmids and DNA vaccines can very effectively convert Th2 immune responses to Th 1. CD4 + CD25 + Foxp3 + T cell proliferation is obvious, which indicates that the recombinant plasmid and DNA vaccine of the invention can inhibit inflammation through up-regulating regulatory T cells. These all confirm that the recombinant plasmid and DNA vaccine provided by the invention have good therapeutic effect on allergic diseases.
Drawings
FIG. 1 is a flow chart of the construction and administration time of AR model mice in one embodiment of the invention.
FIG. 2 is a graph showing the results of plasmid construction and nano-packaging in vitro of pVAX1-Der p2-A20 (pDA) in the example of FIG. 1 according to the present invention. Wherein 2A is a double enzyme digestion result, 2B is a sequencing result, 2C is a nano-packaged electron microscope result, and 2D (I) and 2D (II) are in vitro transfected 293T cells.
FIG. 3 shows the results of expression, purification and immunological activity characterization of recombinant allergen Der p2 of house dust mite in the example of FIG. 1 of the present invention. Wherein 3A is the identification result of recombinant Der p2 expression plasmid, 3B is the induction expression result of recombinant Der p2 protein (M: protein Marker;1: before induction; 2: after induction; 3: supernatant; 4: precipitation), 3C is the affinity chromatography result of recombinant Der p2 protein, 3D is the SDS-PAGE result of recombinant Der p2 protein at the time of eluting peak (M: protein Marker;1: at the beginning of eluting; 2: at the time of eluting peak), 3E is ELISA for detecting the immunogenicity of Der p2 protein (NC: healthy human serum; AR: house dust mite allergic patient positive serum), 3F is Western blot for detecting the allergenicity of Der p2 protein (M: protein Marker;1: healthy human serum; 2: house dust mite allergic patient positive serum).
Fig. 4 is a behavioral modification scoring result for NC, AR, ppDA, PLGA, pDA, pD mice in the embodiment of fig. 1 of the present invention. Where 4A is the nasal grab score of each group of mice and 4B is the sneeze score of each group of mice.
FIG. 5 is a graph (200X) of the staining of the nasal mucosa HE of each group of mice in the example of FIG. 1 of the present invention, and 5A-5F represent the staining results of NC group, AR group, ppDA group, PLGA group, pDA group, pD group, respectively.
FIG. 6 is a graph showing the results of detection of Der p 2-specific antibodies in the serum of each group of mice in the example of FIG. 1 of the present invention, and FIGS. 6A to 6C show IgE antibodies, igG1 antibodies, and IgG2a antibodies, respectively.
FIG. 7 is a graph showing the results of the expression levels of cytokines in the supernatants of spleen lymphocytes of mice of each group in the example of FIG. 1 of the present invention, and 7A-7F represent the expression levels of IFN-gamma, IL-4, IL-10, IL-13, TGF-beta 1, TNF-alpha, respectively.
FIG. 8 is a group of mouse spleen mononuclear cells CD4 of the embodiment of FIG. 1 of the present invention + CD25 + Foxp3 + Flow results for T cells.
Detailed Description
The conception, specific structure, and technical effects produced by the present invention will be clearly and completely described in connection with the embodiments below to fully understand the objects, features, and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
1. Experimental method
Female, 6-8 weeks old, 60 Balb/c healthy mice were selected and purchased from medical laboratory animal center in Guangdong province. The animals were treated in the same manner as in Table 1, and were randomly divided into 6 groups of 10 animals each, each divided into a control group (NC), a model group (AR), a nanovaccine treatment group (PpDA), a blank nanotreatment group (PLGA), a naked plasmid 1 treatment group (pDA) and a naked plasmid 2 treatment group (pD). 5 of the mice in each group were used for HE staining with intact nasal cavities and the remaining 5 of each group were used for protein expression assays with nasal mucosa.
Table 1 test animal grouping and treatment conditions
Connecting the target genes Der p2 and A20 together through a linker, inserting the target genes Der p2 and A20 into a target vector pVAX1 in a cloning mode of Eco RI and Not I, finally extracting plasmids through screening, sequencing the plasmids by a sequencing company, and verifying the accuracy of a target recombinant plasmid (pVAX 1-Der p2-A20 eukaryotic expression vector, pDA). Another recombinant plasmid (pVAX 1-Der p2 eukaryotic expression vector, pD) was obtained in the same manner.
Primers were designed based on the coding sequence of the gene (GenBank accession number of Der p2 gene is FM177223.1, and GenBank accession number of A20 gene is KJ 892292.1), and were commissioned to be synthesized by Shanghai Bioengineering Co., ltd. Specific primer information is as follows:
der p2 amplification primers:
upstream primer (SEQ ID No. 1): AGCCGTTTTCGAAGCCAACC;
downstream primer (SEQ ID No. 2): GGATCGATACCGGGAACATCAAC.
A20 amplification primers:
upstream primer (SEQ ID No. 3): CGGAATTCCACCATGGCTGAACAAGT;
downstream primer (SEQ ID No. 4): CCGGATATCTTAGCCATACATCTGC.
A PLGA-pVAX1-Der p2-A20 (PpDA) DNA vaccine is prepared by adopting a ultrasonic emulsification method and taking polylactic acid-glycolic acid copolymer (PLGA) as an encapsulating material. 100mg of PLGA was weighed and dissolved in 900. Mu.L of methylene chloride and 100. Mu.L of acetone to form an organic phase; 100. Mu.L of pVAX1-Der p2-A20 naked plasmid 1 (concentration 10. Mu.g/. Mu.L) dissolved in sterile ultra pure water was taken as an inner aqueous phase; mixing the organic phase and the internal water phase, and performing ultrasonic crushing in ice water bath for 25 times (the power is 40W, and the intermittent time/continuous working time is 6s/6 s) to form milky colostrum; 2mL of a 2% aqueous solution of polyvinyl alcohol (PVA) was slowly added dropwise to the colostrum as an external aqueous phase, and ultrasound was again performed under the same conditions to form a multiphasic multiple emulsion. Adding 50mL of deionized water into the multiphase compound emulsion, and stirring (or rotary evaporation) at room temperature for 4 hours until the organic solvent is completely volatilized; centrifuging at 4deg.C and 10000rpm for 20min, collecting precipitate, and washing with sterile water for 3 times; freeze-drying, and storing at-80deg.C.
The BL21 engineering bacteria containing pET28a-Der p2 plasmid is induced and expressed in large quantity by isopropyl thiogalactoside (IPTG), the expression product exists in the form of recombinant protein inclusion body, and after washing and dissolving the inclusion body, the expression product passes through Ni 2+ The recombinant protein Der p2 was purified by affinity chromatography, filtered through ultrafiltration membrane, and the purified product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The immunological characteristics of Der p2 recombinant proteins were analyzed by ELISA and Western blot using positive serum from house dust mite allergic patients.
FIG. 1 is a flow chart of the construction and administration time of AR model mice of the present invention. As shown in fig. 1, the preparation process of the AR model is specifically as follows:
mice were sensitized on days 0, 7, and 14, respectively, with AR, ppDA, PLGA, pDA, and pD groups each injected intraperitoneally with 100 μg Der p2 recombinant protein and 15 μg Cholera Toxin (CT), and NC groups were replaced with PBS. 1 week after the end of sensitization, the PpDA group, the PLGA group, the pDA group, and the pD group were subjected to nasal drip immunotherapy with PpDA nanovaccine (containing 100. Mu.g of pVAX1-Der p2-A20 plasmid), blank PLGA nanoparticles, 100. Mu.g of pVAX1-Der p2-A20 plasmid, and 100. Mu.g of pVAX1-Der p2 plasmid, respectively, 1 nasal drip every 3 days for 5 total treatments. Mice were challenged with Der p2 recombinant protein (200 μg) separately by nasal drops 1 time a day for 3 consecutive days after the last treatment, and sacrificed 24h after the end of the challenge. The PpDA nano vaccine used in the scheme is the PBS-dissolved nano particle for the DNA vaccine prepared by 1.3DNA vaccine PLGA-pVAX1-Der p2-A20 (PpDA), can be directly administrated by nasal drip, directly acts on the pathological part of allergic rhinitis, and does not need intramuscular injection.
2. Experimental results
The amplified Der p2 and A20 full-length genes are sequenced and then subjected to sequence comparison by DNASsist 1.0 software, and the result shows that the obtained Der p2 gene and A20 gene are completely consistent with the Der p2 and A20 full-length coding sequences published in Genbank. FIG. 2 is a graph showing the results of construction of the recombinant plasmid of interest and nano-packaging. Wherein, 2A is the double enzyme digestion result, and 2B is the sequencing result. As shown in 2A and 2B, the size of a band generated by double enzyme digestion of the target recombinant plasmid pVAX1-Der p2-A20 is consistent with that of a target gene fragment; the identified positive clone bacteria are sequenced, and the obtained recombinant gene pVAX1-Der p2-A20 is proved to have homology comparison between the Der p2 gene and the A20 gene in the gene and the sequences in Genbank, and the homology between the cloned sequences and the published full-length sequences is 100%, which indicates that the eukaryotic expression plasmid pVAX1-Der p2-A20 is successfully constructed.
FIG. 2 is a graph of the results of construction of the recombinant plasmid of interest and nano-packaging, wherein 2C is the electron microscopy result of nano-packaging, and 2D (I) and 2D (II) are the results of in vitro transfection of 293T cells. As shown in 2C, the PpDA nano DNA vaccine is elliptical and smooth particles under a scanning electron microscope, and has the average particle diameter of 300-500nm, uniform size and good dispersibility. The uptake rates of the nanovaccine into 293T cells are shown as 2D (I) and 2D (II) under confocal microscopy.
FIG. 3 shows the results of expression, purification and immunological activity characterization of recombinant allergen Der p2 from house dust mites. Wherein 3A is the identification result of recombinant Der p2 expression plasmid, 3B is the induction expression result of recombinant Der p2 protein (M: protein Marker;1: before induction; 2: after induction; 3: supernatant; 4: precipitation), 3C is the affinity chromatography result of recombinant Der p2 protein, 3D is the SDS-PAGE result of recombinant Der p2 protein at the time of eluting peak (M: protein Marker;1: at the beginning of eluting; 2: at the time of eluting peak), 3E is ELISA for detecting the immunogenicity of Der p2 protein (NC: healthy human serum; AR: house dust mite allergic patient positive serum), 3F is Western blot for detecting the allergenicity of Der p2 protein (M: protein Marker;1: healthy human serum; 2: house dust mite allergic patient positive serum). As shown in figure 3, the experiment successfully constructs a prokaryotic expression plasmid pET28a (+) -Der p2, converts the plasmid into E.coli BL21 to induce expression, and after affinity chromatography purification, SDS-PAGE shows that target protein is obtained, ELISA and Western blot verify that the target protein can be combined with specific IgE of positive serum of house dust mite allergic patient, and the Der p2 recombinant protein after induction expression purification has immunological activity.
After the last nasal challenge, the mice were observed for behavioral changes over 30min and the number of nasal grabs and sneezes was recorded. The scoring criteria were:
grabbing a nose: the number of the grabbers is more than 3, the number of frequent grabbers (10-20 times/min) is 2, the number of the mild grabbers is 1, and the number of the non-grabbers is 0;
sneeze: more than 11 sneezes are 3 points, 4 to 10 are 2 points, 1 to 3 are 1 point, and no sneeze is 0 point.
And recording total scores by adopting an superposition method, wherein the total score is more than 5 scores, and the successful molding is indicated.
Fig. 4 is the results of behavioral change scores for NC, AR, ppDA, PLGA, pDA, pD mice. Where 4A is the nasal grab score of each group of mice and 4B is the sneeze score of each group of mice. As shown in FIG. 4, NC group was occasionally grabbed with nose, AR group was grabbed with nose and sneeze, and the scores were all > 5 points, indicating successful modeling.
The allergic symptoms of the PpDA, pDA and pD groups were significantly reduced compared to the AR group, with the most significant reduction with the PpDA group and no significant change in the PLGA group; the symptom scores of PpDA, pDA, and pD groups were significantly lower than the AR group, with the decrease in symptom score of PpDA group being more pronounced, and the PLGA group being not significantly altered from the AR group, both scores of PpDA group being lower than both scores of pD group, particularly sneezing score. This suggests that PpDA nanovaccine significantly improved allergic symptoms in allergic rhinitis mice, while simultaneous expression of Der p2 and a20 was more pronounced in improvement of allergic symptoms than Der p2 alone.
After the last excitation for 24 hours, the mice are killed by anesthesia and neck breaking, the complete nasal cavities (n=5) of the mice are taken out and fixed in 4% paraformaldehyde for 24 hours, then a proper amount of vinegar-hydrochloric acid formaldehyde decalcification solution is prepared, decalcification is carried out for 6 hours, and then gradient dehydration and paraffin embedding of different concentrations of ethanol are carried out, conventional sections with the thickness of 5um are carried out, and observation and photographing are carried out under a hematoxylin-eosin staining (HE staining) rear-view mirror. FIG. 5 is a graph (200X) showing the staining of the nasal mucosa HE of each group, and 5A-5F represent the staining results of NC group, AR group, ppDA group, PLGA group, pDA group, and pD group, respectively. As shown in fig. 5, the nasal mucosa of the mice in AR group and PLGA group is edema, vasodilation, gland hyperplasia, and massive eosinophil infiltration can be seen in the intrinsic layer; the nasal mucosa of NC mice did not show significant inflammatory response; the nasal mucositis response was significantly reduced in the PpDA, pDA, and pD groups compared to the AR group, with the most significant reduction in the PpDA group, which in turn was more pronounced than in the pD group. The above results indicate that simultaneous expression of Der p2 and a20 in the PpDA nanovaccine may have a more pronounced therapeutic effect on allergic rhinitis mice than PpD nanovaccine alone.
About 1mL of blood was collected from the retrobulbar venous plexus of the mice, and Der p2 allergen-specific IgE, igG1 and IgG2a were detected in the serum by indirect ELISA. The recombinant Der p2 allergen was coated at 5. Mu.g/mL, blocked at 4℃overnight with 3% BSA/PBS. Mouse serum (IgE, 1:5,IgG1,1:2000 and IgG2a, 1:1000) was diluted with 1% BSA/PBST and incubated at 37℃for 1h. After washing the plates, secondary antibodies (IgE, igG1 and IgG2 a) were added and incubated for 1h at 37 ℃. The plate is washed again, TMB substrate is added and developed for 10-15min in dark. After termination of the substrate reaction and immediately reading the OD at 450 nm.
FIG. 6 is a graph showing the results of Der p 2-specific antibodies in the serum of each group of mice in the example of FIG. 1 of the present invention, and FIGS. 6A-6C show IgE antibodies, igG1 antibodies, and IgG2a antibodies, respectively. As shown in FIG. 6, the serum levels of Der p 2-specific IgE (Der p 2-sIgE) were significantly higher in both the AR group and the PLGA group compared to the NC group, indicating successful modeling. Der p2-sIgE was significantly reduced in serum from the PpDA, pDA and pD groups compared to the AR group, with the reduction being more pronounced with the PpDA group, whereas the pDA group was significantly reduced compared to the pD group. Both IgG1 and IgG2a levels were significantly elevated in the serum of the PpDA group and the pDA group compared to the AR group, with the elevation being more pronounced with the PpDA group, and also with the pDA group compared to the pD group; the serum IgG1 and IgG2a levels were not significantly different in the PLGA group compared to the AR group, the PpDA group was more significantly elevated, and the pDA group was also significantly elevated compared to the pD group. The detection results of the three allergen-specific antibodies are combined to show that the PpDA nanovaccine has a certain therapeutic effect, and the simultaneous expression of Der p2 and A20 is very remarkable in improving the therapeutic effect compared with the independent Der p 2.
Spleen lymphocytes of mice were isolated under aseptic conditions, cultured in RPMI1640 medium for 72h in a constant temperature incubator, and the supernatant was collected by centrifugation. The levels of IFN-gamma, IL-4, IL-10, IL-13, TGF-beta 1 and TNF-alpha in the supernatants were determined by ELISA, and the procedures were strictly followed by kit instructions.
FIG. 7 is a graph showing the results of the expression levels of cytokines in the supernatants of spleen lymphocytes of each group of mice, and 7A-7F represent the expression levels of IFN-gamma, IL-4, IL-10, IL-13, TGF-beta 1, TNF-alpha, respectively. As shown in FIG. 7, the IL-4 and IL-13 levels in the spleen cell culture supernatants of the AR group were significantly increased compared to the NC group, indicating successful sensitization. IL-4, IL-13 and TNF- α levels were significantly reduced in splenocyte culture supernatants of PpDA, pDA and pD groups compared to AR groups, IFN- γ, IL-10 and TGF- β1 levels were significantly increased, with the increase in PpDA groups being more pronounced, whereas PLGA groups were not significantly different from AR groups. The PpDA nano vaccine can reduce inflammatory response of allergic rhinitis mice, and can convert Th2 immune response to Th1 direction.
Spleen lymphocytes from mice were isolated under aseptic conditions and the cell concentration was adjusted to 1X 10 6 /mL. The spleen mononuclear cells were first stained with APC Rat Anti-Mouse CD4 and FITC Rat Anti-Mouse CD25 flow antibody for 15min, then fixed with paraformaldehyde for 15min, PBS washed three times, and finally stained overnight with a transmembrane solution containing PE Rat Anti-Mouse eFox 3, and the following day with flow cytometry for detection of CD4 in spleen mononuclear cells + CD25 + Foxp3 + T cell ratio and data were analyzed using Flowjo software. The results are shown in FIG. 8. PpDA group and pDA group CD4 compared to AR group + CD25 + Foxp3 + T cell ratio was increased, with the increase more pronounced with the PpDA group, while the PLGA group was not significantly different from the AR group. Due to CD4 + CD25 + Foxp3 + T cells can regulate the occurrence of inflammation, so the experimental result shows that the PpDA nano vaccine has a certain therapeutic effect.
In summary, the levels of IL-4, IL-13, TNF- α were lower in the PpDA group, and more IL-10, IFN- γ, TGF- β1, and CD4 were present in the PpDA group, as compared to the pDA group + CD25 + Foxp3 + The T cell proportion is higher, which indicates that the PLGA encapsulated pVAX1-Der p2-A20 nanometer DNA vaccine has better effect of treating allergic rhinitis compared with naked plasmid (pVAX 1-Der p 2-A20) by directly dripping nose immunity, thereby having better application prospect and practical value for preventing and treating allergic rhinitis. While pDA groupThe combined behavioral changes, histopathological detection and comparison of allergen specific antibodies and others can also be seen as the synergistic effect of simultaneous expression of Der p2 and a20 compared with the pD group, and the effect of Der p2 on alleviating the symptoms is significantly improved compared with that of Der p2 alone, so that the Der p2-a20 nano DNA vaccine has more outstanding therapeutic effect on treating allergic diseases, particularly allergic rhinitis compared with that of Der p2 nano DNA vaccine alone.
While the invention has been described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes and substitutions may be made therein without departing from the spirit of the invention and that these changes and substitutions are intended in the scope of the invention as defined by the appended claims.
SEQUENCE LISTING
<110> Liu Zhijiang Shenzhen institute of ear, nose and throat
<120> recombinant plasmid, DNA vaccine, and preparation method and application thereof
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<170> PatentIn version 3.3
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<211> 20
<212> DNA
<213> artificial sequence
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agccgttttc gaagccaacc 20
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ggatcgatac cgggaacatc aac 23
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cggaattcca ccatggctga acaagt 26
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Claims (4)
1. A DNA vaccine applied to allergic rhinitis, which is characterized by comprising a recombinant plasmid and a polylactic acid-glycolic acid copolymer as an entrapment material, wherein the recombinant plasmid comprises a target vector pVAX1 and a target gene inserted into the target vector pVAX1, the target genes Der p2 and A20 are connected together through a linker, the GenBank accession number of Der p2 is FM177223.1, and the GenBank accession number of A20 is KJ892292.1;
wherein, the DNA vaccine is prepared by the following method:
dissolving polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase;
mixing the solution of the recombinant plasmid with the organic phase to form colostrum;
mixing the primary emulsion with a polyvinyl alcohol aqueous solution to form a multiphase emulsion;
removing the organic solvent from the multiphase emulsion to form microspheres.
2. The method for preparing the DNA vaccine according to claim 1, comprising the steps of:
dissolving polylactic acid-glycolic acid copolymer in an organic solvent to form an organic phase;
mixing the solution of the recombinant plasmid with the organic phase to form colostrum;
mixing the primary emulsion with a polyvinyl alcohol aqueous solution to form a multiphase emulsion;
removing the organic solvent from the multiphase emulsion to form microspheres.
3. Use of the DNA vaccine of claim 1 in the manufacture of a medicament for the prevention or treatment of allergic diseases.
4. The use according to claim 3, wherein the allergic disease is allergic rhinitis.
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