CN102000330A - Nucleic acid vaccine adjuvant and construction method thereof - Google Patents
Nucleic acid vaccine adjuvant and construction method thereof Download PDFInfo
- Publication number
- CN102000330A CN102000330A CN 201010280672 CN201010280672A CN102000330A CN 102000330 A CN102000330 A CN 102000330A CN 201010280672 CN201010280672 CN 201010280672 CN 201010280672 A CN201010280672 A CN 201010280672A CN 102000330 A CN102000330 A CN 102000330A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- acid vaccine
- flt3l
- plasmid
- vaccine adjuvant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108700001237 Nucleic Acid-Based Vaccines Proteins 0.000 title claims abstract description 33
- 229940023146 nucleic acid vaccine Drugs 0.000 title claims abstract description 33
- 239000012646 vaccine adjuvant Substances 0.000 title claims abstract description 16
- 238000010276 construction Methods 0.000 title abstract description 5
- 239000013612 plasmid Substances 0.000 claims abstract description 30
- 101710162577 Fms-related tyrosine kinase 3 ligand protein Proteins 0.000 claims abstract description 26
- 230000002708 enhancing effect Effects 0.000 claims abstract description 7
- 230000007969 cellular immunity Effects 0.000 claims abstract 6
- 108020004414 DNA Proteins 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 102000036639 antigens Human genes 0.000 abstract description 20
- 108091007433 antigens Proteins 0.000 abstract description 20
- 102100020715 Fms-related tyrosine kinase 3 ligand protein Human genes 0.000 abstract description 19
- 239000000427 antigen Substances 0.000 abstract description 19
- 210000004027 cell Anatomy 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 7
- 238000002347 injection Methods 0.000 abstract description 6
- 239000007924 injection Substances 0.000 abstract description 6
- 239000002671 adjuvant Substances 0.000 abstract description 5
- 230000001413 cellular effect Effects 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
- 102100037850 Interferon gamma Human genes 0.000 abstract description 4
- 108010074328 Interferon-gamma Proteins 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 4
- 210000000952 spleen Anatomy 0.000 abstract description 3
- 108010071942 Colony-Stimulating Factors Proteins 0.000 abstract description 2
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 abstract description 2
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 abstract description 2
- 230000004069 differentiation Effects 0.000 abstract description 2
- 210000003958 hematopoietic stem cell Anatomy 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 229940124931 vaccine adjuvant Drugs 0.000 abstract description 2
- 230000024932 T cell mediated immunity Effects 0.000 abstract 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 abstract 1
- 230000035755 proliferation Effects 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 108090000623 proteins and genes Proteins 0.000 description 19
- 230000036039 immunity Effects 0.000 description 15
- 229960005486 vaccine Drugs 0.000 description 12
- 230000003053 immunization Effects 0.000 description 10
- 238000002649 immunization Methods 0.000 description 9
- 230000004044 response Effects 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 5
- 230000000890 antigenic effect Effects 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000011238 DNA vaccination Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000013467 fragmentation Methods 0.000 description 4
- 238000006062 fragmentation reaction Methods 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 230000002045 lasting effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101001052849 Homo sapiens Tyrosine-protein kinase Fer Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010064912 Malignant transformation Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 102100024537 Tyrosine-protein kinase Fer Human genes 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 230000000139 costimulatory effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 230000008073 immune recognition Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 239000000568 immunological adjuvant Substances 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000036212 malign transformation Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
Images
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及医药生物学技术领域。Flt3L为集落刺激因子家族成员之一，可刺激CD34+造血干细胞的增殖和分化。本发明的目的在于提供一种具有细胞免疫增强活性的核酸疫苗佐剂及其构建方法，本发明的核酸疫苗佐剂是一种基于Flt3L设计的用于增强核酸疫苗细胞免疫效果的质粒。本发明经体内试验证明，该质粒与核酸疫苗同时注射具有有效地诱导细胞免疫反应的能力，表现为抗原特异性脾脏IFN-γ阳性细胞增多和脾脏CD8阳性细胞杀靶能力增强。因此，本发明的疫苗佐剂可以添加到现有核酸疫苗制剂内混合注射，从而显著促进核酸疫苗诱导的细胞免疫反应，作为一种新型的细胞免疫佐剂。The invention relates to the technical field of medical biology. Flt3L is a member of the colony-stimulating factor family, which can stimulate the proliferation and differentiation of CD34+ hematopoietic stem cells. The object of the present invention is to provide a nucleic acid vaccine adjuvant with cellular immunity enhancement activity and its construction method. The nucleic acid vaccine adjuvant of the present invention is a plasmid designed based on Flt3L for enhancing the cellular immunity of nucleic acid vaccines. In vivo experiments prove that the simultaneous injection of the plasmid and the nucleic acid vaccine has the ability to effectively induce cellular immune response, which is manifested by the increase of antigen-specific spleen IFN-γ positive cells and the enhanced ability of spleen CD8 positive cells to kill targets. Therefore, the vaccine adjuvant of the present invention can be added to the existing nucleic acid vaccine preparations for mixed injection, thereby significantly promoting the cellular immune response induced by the nucleic acid vaccine, as a new type of cellular immune adjuvant.
Description
Technical field
The present invention relates to the medicine bioengineering field that learns a skill.In the art, the invention particularly relates to the immunocompetence enhancement techniques field of nucleic acid vaccine.
Background technology
Nucleic acid vaccine is meant that the plasmid vector that contains certain antigenic protein gene sequence is as vaccine; directly import in the zooblast; by host's re-recording system synthetic antigen albumen, induce the host to produce immunne response, thereby make the host obtain corresponding immunoprotection this antigen protein.Because nucleic acid vaccine can induce body to produce comprehensive immunne response; and the pathogen to different subtype has the effect of resisting that intersects; have simultaneously series of advantages such as immune protective effect is lasting, safe, convenient for production again, so be considered to the third generation vaccine after attenuation, inactivated vaccine and genetic engineering subunit vaccine.The advantage of nucleic acid vaccine mainly contains: 1, not only can induce the protection antibody at conservative antigen to produce; and CTL immunity that can the simultaneous excitation body, the prevention of the disease of dependent cells immune clearance cause of disease such as object disease of viral infection is also just more effective like this.And after the gene vaccine inoculation, antigen is consistent with the expression of virus antigen in the natural viral infection process in intracellular expression, can make antigen give immune recognition system with the processed back of the form of nature submission.This is necessary for the generation that the conformation type epitope brings out neutrality antibody; 2, preparation is easy, the preparation of time saving and energy saving gene vaccine only needs the gene of coding for antigens is designed and clones, do not need at vivoexpression and protein purification, and exogenous gene is easier to the clone and advances expression vector, can increase in a couple of days and purification it, particularly when epidemic diseases is broken out, can develop new vaccine fast to those.3, immunne response is more lasting, and bibliographical information 15 months available PCR after intramuscular injection detect the existence of exogenous plasmid dna.Experimental results show that in injection and still can detect a considerable amount of expression of exogenous gene in back 19 months.Exogenous gene and express to continue exist like this make that the memory of special immunological memory cell is reinforced in the body, thereby the protective immunity that body is obtained exists more lastingly.4, different strain cross protection of the same race this be one of great advantage of gene vaccine.In preparation during gene vaccine, can be by the entrained target gene of expression vector be transformed, thus select antigenic determinant.The nucleotide sequence of coding conservative protein of selecting a certain pathogen is as gene vaccine, because of it can not make a variation, thus can be to drift or novel generation cross immunity with a kind of cause of disease, thus play immanoprotection action.5, can be used for that treatment and prevention of tumour CTL replys also is effective removing approach that body kills cancerous tumor cell.If can find the key protein in the malignant transformation of cells process, just can prepare the CTL vaccine of tumor.After the inoculation of this gene vaccine, can bring out body and produce the CTL immunne response, immune surveillance is carried out in cancerating of pair cell, and the cell of canceration is produced immunne response, thereby provides strong modern weapon for the prevention and the immunization therapy of cancer.
Yet dna vaccination also exists tangible deficiency, and promptly dna vaccination stimulates a little less than the immunoreation that ability that body produces immunne response often causes than the conventional vaccine inoculation, and this just gives the new challenge of having researched and proposed of dna vaccination.Therefore, new vaccine adjuvant has become the research focus that receives much attention now.Immunological adjuvant is meant with antigen and uses simultaneously or in advance, can promote, prolongs or strengthen the material to the vaccine antigen specific immune response.At present most popular in the preparation of animal vaccine is oily adjuvant and aluminum salt adjuvant etc., and mainly contain cytokine, costimulatory molecules, CpG DNA and liposome (Donnelly as the adjuvant of dna vaccination, JJ, Wahren, B, and Liu, MA.DNA vaccines:progress and challenges.J Immunol., 2005,175:633-639.).
Mice FMS sample tyrosine kinase 3 parts (Flt3L) are one of colony stimulating factor family member, belong to tyrosine kinase receptor, can stimulate the propagation and the differentiation of CD34+ hematopoietic stem cell, and induce the maturing dendritic cell of specific subgroup.Discover, by injection Flt3L the antigen specific immune reaction is significantly strengthened, and significantly increase quantity and the frequency of DC, Flt3L can have synergism with the various kinds of cell factor, be a kind of good DC amplification agent, in toy and people's body, reach external experimentizing and all obtained good effect (Pulendran B.Modulatingvaccine responses with dendritic cells and Toll-like receptors.Immunol Rev.2004; 199:227-50).And inducing of antigen-specific immune response depends on that to a great extent body is to the processing of this antigen molecule and the efficient of offering.Based on the important function of DC in genetic immunization,, then may be beneficial to the effect of enhancing gene immunity if the APC that recruitment comprises DC to position, target antigen place, increases antigenic picked-up and offers.
Summary of the invention
The object of the present invention is to provide a kind of nucleic acid vaccine adjuvant and construction method thereof with cellular immunization enhanced activity.
The present invention imagines Flt3L as a kind of nucleic acid vaccine adjuvant molecule, the present invention has obtained one section sequence that lacks the part base in clone Flt3L gene process, prove that by experiment this sequence has the effect of raising and activate DC equally, do not see the relevant report of this sequence at present.
The present invention has designed a kind of plasmid pcDNA.3.1-Flt3L of the Flt3L of coding sequence, and this plasmid can efficiently express Mus Flt3L in vivo and in vitro.Through being total to the injecting immune mice with the hepatitis B surface antigen nucleic acid vaccine, we find to improve especially to nucleic acid vaccine immunity effectiveness by this plasmid, and the enhancing of cell immune response plays an important role, therefore, described plasmid and nucleic acid vaccine are injected altogether and be can be used as a kind of new nucleic acid vaccine immunity effectiveness Enhancement Method.
The invention provides a kind of nucleic acid vaccine adjuvant with cellular immunization enhanced activity, it is characterized in that this nucleic acid vaccine adjuvant is the plasmid that contains DNA sequence that can effective expression Flt3L gene splicing body molecule, DNA sequence wherein is shown in SEQ ID NO:4.
Above-mentioned plasmid is pcDNA.3.1 (+), and the present invention also can substitute with other eukaryon expression plasmids such as pVAX1 or pEZX.Plasmid also can substitute with adenovirus, slow virus etc.
The present invention also provides the construction method of above-mentioned nucleic acid vaccine adjuvant, may further comprise the steps: A) design PCR primer is as follows, adopt the extractive genomic DNA of human peripheral blood mononuclear cell to carry out PCR, the DNA sequence of the Flt3L gene splicing body molecule that obtains encoding is shown in SEQ ID NO:4;
B) DNA sequence with above-mentioned coding Flt3L gene splicing body molecule is cloned into plasmid.
Technical scheme particular content of the present invention is as follows:
1, the present invention at first according to internet database www.pubmed.com information searching the Flt3L molecular sequences of mice (GenBank:EU274583.1 is shown in SEQ ID NO:1), at its flanking gene group coding sequential design PCR primer (upstream and downstream is added Xbal I and EcoR I restriction enzyme site respectively), adopt the extractive genomic DNA of human peripheral blood mononuclear cell to carry out PCR, the clone is as follows with the PCR primer:
Forward primer: 3 ' cgggatcc Atg aca gtg ctg gcg cca gcc, 5 ' (shown in SEQ ID NO:2)
Downstream primer: 5 ' gctctagaggg atg gga ggg gag ggg cac c, 3 ' (shown in SEQ ID NO:3)
Thereby obtained comprise Flt3L molecule encoding sequence dna fragmentation (shown in SEQ ID NO:4,585bp).
2, the present invention is connected into above-mentioned clone's the dna fragmentation that comprises the pcDNA.3.1-Flt3L coded sequence (as shown in Figure 1) in the polyclone zone available from the commercialization plasmid pcDNA.3.1 (+) of American I nvitrogene company, adopts Xbal I and EcoR I restriction enzyme site.The polyclone enzyme action site of this insertion sequence flank still keeps, and can be used to insert antigen sequence does not influence its effective expression.Described Flt3L expression vector: the pcDNA.3.1-Flt3L that is.
3, plasmid of the present invention has following attribute through the zoopery checking:
(1) this plasmid can efficiently increase intracellular Flt3L developed by molecule level.
(2) this plasmid can be total to immunity and not influence latter's antigen expressed with various nucleic acid vaccines.
(3) this plasmid and hepatitis B surface antigen nucleic acid vaccine are injected mice altogether, can induce very strong antigenic specificity cell immune response, more traditional nucleic acid vaccine is significantly increased.But the influence of antagonist titre is not remarkable.
(4) in the experiment of heavy dose of this carrier of immunity repeatedly, any unusual allergy or autoimmune phenomena do not appear in laboratory animal.
Description of drawings
Fig. 1: pcDNA3.1-Flt3L plasmid ideograph
With the polyclone zone that Flt3L inserts pcDNA3.1 (+) carrier by Xbal I and EcoR I site, obtain to express the carrier of Flt3L.Green area inserts the site for the Flt3L expressed sequence among the figure, and the multiple clone site beyond the green area still keeps, and can be used for the antigen encoding sequence and inserts.This plasmid contains ammonia benzyl mycin resistant gene.
Fig. 2: immune efficacy in the body after pcDNA3.1-Flt3L plasmid and the common immunity of pVAX1-HBsAg nucleic acid vaccine is detected antibody titer
Fig. 3: immune efficacy in the body after pcDNA3.1-Flt3L plasmid and the common immunity of pVAX1-HBsAg nucleic acid vaccine is detected IFN-γ secretion
The specific embodiment
Below in conjunction with drawings and Examples the present invention is described in detail, but embodiments of the invention only are used to the present invention is described and are not used in restriction protection scope of the present invention.
The structure of embodiment 1::pcDNA3.1-Flt3L plasmid
We at first according to the internet database information searching Flt3L molecular sequences (GenBank:EU274583.1 of mice, www.pubmed.com), at its flanking gene group coding sequential design PCR primer (upstream and downstream is added Xbal I and EcoR I restriction enzyme site respectively), adopt the extractive genomic DNA of human peripheral blood mononuclear cell to carry out PCR, Flt3L molecular sequences and clone use PCR primer such as following table.Thereby obtained the dna fragmentation that comprises Flt3L molecule encoding sequence.
We are connected into above-mentioned clone's the dna fragmentation that comprises the pcDNA.3.1-Flt3L coded sequence (Fig. 1) in the polyclone zone available from the commercialization plasmid pcDNA.3.1 (+) of American I nvitrogene company, adopt Xbal I and EcoR I restriction enzyme site.The polyclone enzyme action site of this insertion sequence flank still keeps, and can be used to insert antigen sequence does not influence its effective expression.Described Flt3L expression vector: the pcDNA.3.1-Flt3L that is.
Embodiment 2:pcDNA3.1-Flt3L plasmid and pVAX1-HBsAg nucleic acid vaccine cotransfection situation lower body appearance Danone power detect.
Utilize mice as experimental animal model, again to mice shank injection pcDNA3.1-Flt3L plasmid, the DC that adopts SABC to analyze injection site after 24 hours raises situation, finds after the immunity of pcDNA3.1-Flt3L plasmid and the matched group contrast, and immune group has significant DC to raise and activate.
Embodiment 3: immune efficacy in the body after pcDNA3.1-Flt3L plasmid and the common immunity of pVAX1-HBsAg nucleic acid vaccine is detected.
With pcDNA3.1-Flt3L with 0.1mg/ only with pVAX 1-HBsAg with 0.1mg/ dosage immunity 6-8 C57BL/6 mice in age in week, immunity is 3 times altogether, 2 weeks of interval, mix incomplete Freund's adjuvant booster immunization once with HBsAg recombiant protein (10 μ g/ only) the 6th weekend, the humoral immunization aspect adopts ELISA to detect its antibody titer (as shown in Figure 2); The cellular immunization aspect, adopt the ELISPOT method to detect the IFN-γ secretion situation of spleen t-cell, found that (as shown in Figure 3), pcDNA3.1-Flt3L with 0.1mg/ only with pVAX1-HBsAg with the only common immunity of 0.1mg/ after effective inducing antigen-specific ctl response, IFN-γ secretion positive cell significantly increases, the target cell killing-efficiency significantly improves, and prompting antigenic specificity cellular immunization obtains obviously to strengthen.
Table 1, experiment grouping, immunizing dose, immunization ways
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102806724A CN102000330B (en) | 2010-09-14 | 2010-09-14 | Nucleic acid vaccine adjuvant and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102806724A CN102000330B (en) | 2010-09-14 | 2010-09-14 | Nucleic acid vaccine adjuvant and construction method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102000330A true CN102000330A (en) | 2011-04-06 |
| CN102000330B CN102000330B (en) | 2012-08-29 |
Family
ID=43808200
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102806724A Expired - Fee Related CN102000330B (en) | 2010-09-14 | 2010-09-14 | Nucleic acid vaccine adjuvant and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102000330B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333095A (en) * | 2013-06-26 | 2013-10-02 | 中国人民解放军第二军医大学 | Adipamide D-hydroxyproline derivative and application thereof as nucleic acid vaccine adjuvant |
| CN106999573A (en) * | 2014-12-02 | 2017-08-01 | 国立大学法人北海道大学 | Adjuvant composition |
| CN117100865A (en) * | 2023-08-23 | 2023-11-24 | 重庆理工大学 | NgR and PirB as immunotherapeutic targets for amelioration of post-traumatic stress disorder-like symptoms |
| CN120789238A (en) * | 2025-09-18 | 2025-10-17 | 国科大杭州高等研究院 | LDLR intervention adjuvant for promoting antibody production and enhancing vaccine effect and application |
-
2010
- 2010-09-14 CN CN2010102806724A patent/CN102000330B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 《中国免疫学杂志》 20081231 蓝佳明 MIP-1alpha和Flt3l基因佐剂联合应用对HPV16E7 DNA疫苗的免疫增强作用 , 2 * |
| 《河北医科大学学报》 20081231 蓝佳明 Flt3l与肿瘤关系的研究进展 , 2 * |
| 《现代免疫学》 20081231 刘春燕 Flt3L与CCL5细胞因子佐剂对HBc抗原DNA疫苗特异性免疫应答的促进作用 , 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333095A (en) * | 2013-06-26 | 2013-10-02 | 中国人民解放军第二军医大学 | Adipamide D-hydroxyproline derivative and application thereof as nucleic acid vaccine adjuvant |
| CN103333095B (en) * | 2013-06-26 | 2015-06-24 | 中国人民解放军第二军医大学 | Adipamide D-hydroxyproline derivative and application thereof as nucleic acid vaccine adjuvant |
| CN106999573A (en) * | 2014-12-02 | 2017-08-01 | 国立大学法人北海道大学 | Adjuvant composition |
| CN106999573B (en) * | 2014-12-02 | 2021-10-19 | 株式会社先端免疫疗法研究所 | Adjuvant composition |
| CN117100865A (en) * | 2023-08-23 | 2023-11-24 | 重庆理工大学 | NgR and PirB as immunotherapeutic targets for amelioration of post-traumatic stress disorder-like symptoms |
| CN120789238A (en) * | 2025-09-18 | 2025-10-17 | 国科大杭州高等研究院 | LDLR intervention adjuvant for promoting antibody production and enhancing vaccine effect and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102000330B (en) | 2012-08-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7663622B2 (en) | Broad spectrum anti-influenza vaccine immunogens and uses thereof | |
| Coban et al. | Molecular and cellular mechanisms of DNA vaccines | |
| JP6153473B2 (en) | Nucleic acid molecule encoding hepatitis B virus core protein and vaccine comprising the same | |
| US10799572B2 (en) | Consensus antigen constructs and vaccines made therefrom, and methods of using the same to treat malaria | |
| JP6117781B2 (en) | Cross-protective arenavirus vaccines and methods for their use | |
| CN102000330A (en) | Nucleic acid vaccine adjuvant and construction method thereof | |
| Pretorius et al. | A heterologous prime/boost immunisation strategy protects against virulent E. ruminantium Welgevonden needle challenge but not against tick challenge | |
| RU2012149036A (en) | PARAPOXVIRUS VECTORS CONTAINING THE RAGES VIRUS ANTIGEN | |
| Henke et al. | Co-expression of interleukin-2 by a bicistronic plasmid increases the efficacy of DNA immunization to prevent influenza virus infections | |
| CN107286245B (en) | PD-L1 and PD-L2 recombinant proteins and uses thereof | |
| WO2020232254A1 (en) | Attenuated yellow fever virus and uses thereof for the treatment of cancer | |
| KR101863335B1 (en) | DNA vaccine for prevention and treatment of fish viral hemorrhagic septicemia | |
| CN101972476B (en) | DNA vaccine adjuvant using Micro RNA-155 and construction method thereof | |
| CN111118063B (en) | FAP alpha and survivin-based DNA and application thereof in preparation of tumor vaccine | |
| Kumar et al. | DNA vaccine: a modern biotechnological approach towards human welfare and clinical trials | |
| Webster | Potential advantages of DNA immunization for influenza epidemic and pandemic planning | |
| CN102038951A (en) | Nucleic acid vaccine adjuvant designed based on MCP-1 and construction method thereof | |
| US12109258B2 (en) | Synthetic malaria immunogens, combinations thereof, and their use to prevent and treat malaria infections | |
| US11179458B2 (en) | Immunogenicity of an optimized synthetic consensus DNA vaccine for porcine epidemic diarrhea virus | |
| WO2013083287A1 (en) | Hpv derived polynucleic acids for therapy | |
| CN102018964A (en) | Three-plasmid co-immune vaccine system capable of breaking immune tolerance and preparation method thereof | |
| Kebabçıoğlu et al. | Next Generation Vaccines | |
| RU2843610C1 (en) | Polynucleotide vaccine and method of jet needle-free delivery thereof into body | |
| CN102838679A (en) | Preparation and application of Her2-neu antigen positive tumor therapeutic vaccine | |
| CN115838762A (en) | CCHFV recombinant eukaryotic expression vector pVAX1-CCHFV-Gc, construction method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120829 Termination date: 20150914 |
|
| EXPY | Termination of patent right or utility model |