CN108159410A - Dengue fever virus vaccine - Google Patents
Dengue fever virus vaccine Download PDFInfo
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- CN108159410A CN108159410A CN201810034931.1A CN201810034931A CN108159410A CN 108159410 A CN108159410 A CN 108159410A CN 201810034931 A CN201810034931 A CN 201810034931A CN 108159410 A CN108159410 A CN 108159410A
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- 206010012310 Dengue fever Diseases 0.000 title claims abstract description 47
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Abstract
The present invention provides a kind of dengue fever virus vaccines, the present invention from reporting that less dengue fever virus T cell immunology sets about at present, vaccine is built using simian adenoviral vector to overcome the limitation in human-like adenovirus vector practice, successfully construct II type dengue fever virus Ad carrier bacterin C9 NS1, the quick flush end clone technology clone PCR products of the carrier, the double digestion operation of plasmid and target fragment is avoided, it is more convenient quick.The C-terminal of carrier expression mountain fusion protein has the affinitive layer purification that histidine tag facilitates recombinant protein, and histidine-tagged protein is smaller, and influence is nearly free from the activity and function of expressing albumen.The present invention lays the foundation from the immune function of the vaccine of many aspects overall merit 1 albumen sources of dengue fever virus NS further to research and develop efficient spectrum dengue fever gene vaccine.
Description
Technical field
The present invention relates to a kind of vaccines, and in particular to a kind of dengue fever virus vaccine.
Background technology
Dengue fever virus (Dengue virus) is small-sized flavivirus, belongs to Flavivirus, is a kind of acute biography of mosquito matchmaker
The pathogen caught an illness leads to dengue fever, the diseases such as dengue fever heat and dengue shock synthesis increasing.Usually by biting people's on daytime
Aedes aegypti and aedes albopictus are propagated.Dengue fever virus can cause a series of clinical symptoms, lose blood including what is be in peril of one's life
Property shock syndrome and the more rare oxyhepatitis to decline with liver with encephalopathy.Infect dengue fever virus gently then fever suddenly, play
Strong myalgia, osteoarthrosis pain, heavy then extensive bleeding, rapid shock.
Dengue fever virus is tunicary sub-thread, positive chain RNA virus, including 4 different serotypes, dengue fever virus base
Because of group leader 10-11kb, encode 3 kinds of structural proteins (C, PrM, E) and 7 kinds of non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a,
NS4b、NS5).It is still not clear currently based on the immunologic mechanism of NS1 albumen, thinks that humoral immunity accounts for main function mostly.NS1 makees
For a kind of protected protein, it is considered holding promise as the antigen of research dengue fever gene vaccine.Preferably dengue vaccine should be
Primary immunization can prevent the virus infection of 4 serotype simultaneously, and the immune response between 4 serotypes is balanced, and there is no add
The potential risk of weight disease.In addition, ideal dengue virus attenuated live vaccine cannot generate too high virus in subject's body
Titre, but can replicate in vivo, stimulation body generates enough immune responses.
Invention content
Based on this, the present invention provides a kind of dengue fever virus vaccines.
The technical solution that the present invention takes is as follows：
1. a kind of dengue fever virus vaccine, the vaccine is genetic vaccine, with the adenovirus of E1 gene orders missing
Carrier H5 is vector construction, and contains dengue fever virus NS1 protein gene.
Preferably, immunologic adjuvant is further included.
Preferably, the immunologic adjuvant is Freund's complete adjuvant or incomplete Freund's adjuvant.
Preferably, NS1 protein gene sequence of the dengue fever virus NS1 protein gene for the strain of 2 type virus Hainan of dengue fever
Row, sequence is as shown in SEQ NO.1.
2. adenovirus vector-NS1 recombinant proteins, using the adenovirus vector H5 of E1 gene orders missing as carrier, importing is stepped on
Acquisition is expressed after removing from office fever virus NS1 protein gene.
3. a kind of polyclonal antibody by above-mentioned recombinant protein as antigen, is prepared through immune response.
4. one group of immunogenic polypeptide contains 10 by what the NS1 protein gene sequences of 2 type Hainan of dengue fever virus strain synthesized
The polypeptide of amino acid repetitive sequence, it is AGPWHLGKL to repeat peptide sequence.
Dengue vaccine using adenovirus as vector construction can induce the relatively high titre for 4 serotype dengue fever virus
Neutralizing antibody, and 4 serotype antibody levels are balanced.
The beneficial effects of the present invention are：The present invention from reporting that less dengue fever virus T cell immunology at present
Hand builds vaccine to overcome the limitation in human-like adenovirus vector practice, including being made using simian adenoviral vector
A series of unrelated reactions etc., successfully construct II type dengue fever virus Ad carrier bacterins caused by immunocompetence and heterograft
C9-NS1.The quick flush end clone technology clone PCR products of the carrier avoid the double digestion operation of plasmid and target fragment, than
It is quick and easy.The C-terminal of carrier expression mountain fusion protein has the affinitive layer purification that histidine tag facilitates recombinant protein, group
His tag albumen is smaller, and influence is nearly free from the activity and function of expressing albumen.And from many aspects overall merit
The immune function of the vaccine of 1 albumen sources of dengue fever virus NS is established further to research and develop efficient spectrum dengue fever gene vaccine
Fixed basis.It since dengue virus infection is extensive, and is mainly propagated in developing country, the trend of getting worse is to human health
Constitute great threat.From the point of view of preclinical and clinical study results, the prospect of genetic recombination attenuated live vaccine is relatively expected
And it is expected to be approved to list interior in recent years.
Description of the drawings
Fig. 1 analyzes collection of illustrative plates for the upper DENV2NS1 Protein T cell surface antigen IFN γs-ELISPOT of mouse；
Fig. 2 is the NS1 PROTEIN Cs D8 of 26 plant of 4 different serotypes DV+T cell surface antigen conserved sequence；
Fig. 3 is analyzed for T cell antigen epitope sequences conserved features in BALB/C mice experiment；
The functional cell dissolving CD8 of CTL experiments detection C9-NS1 immunized mices in Fig. 4 bodies+T cell.
Specific embodiment
The embodiment of technical solution of the present invention will be described in detail below.Following embodiment is only used for clearer
Ground illustrates technical scheme of the present invention, therefore is only used as example, and is not intended to limit the protection scope of the present invention and limits the scope of the invention.
First, dengue fever virus vaccine is built
1. all adenovirus vectors are purchased from lucky triumphant gene；
The dengue fever 2 that 2.NS1cDNA is logged according to GeneBank (NC_002020.1, sequence is as shown in SEQ NO.1)
The sequence chemical synthesis of type virus Hainan strain；
3. when synthesizing NS1 genes, I sites of Xba, Kozak signals and tissue plasminogen activator (TAP) sequence quilt
5 ' ends are added to, 2 terminators and I sites of Xba are added to 3 ' ends, so that subsequent experimental operates；It is specific as follows：(1) it is sick
Malicious Total RNAs extraction；(2) the RT-PCR amplifications of gene cDNA；(3) PCR product agarose gel electrophoresis is observed；(4) gram of gene
Grand and sequence verification；
4. the NS1 genetic fragment admittances of above-mentioned synthesis are entered what E1 gene orders lacked by Direct Cloning and screening technique
By PCR amplification by NS1 gene outcomes, the segment of acquisition is connected in H5 carriers by human adenovirus vector H5, by conversion,
Double digestion and sequence verification；
5. being proliferated in HEK293 cells and obtaining H5-NS1 recombinant viral proteins, operation is as follows：(1) by above-mentioned recombination
Human adenovirus vector is transfected into KEK293 cells, is collected by the way that whether fluoroscopic examination is transfected after successfully (2) cultivate 72h；(3) it is sharp
The relative molecular mass of expression albumen, expression-form and optimum inductive condition are examined with SD-PAGE, and utilizes Western blot
Identify target protein；(4) recombinant protein purifies.
Sequence is repeated containing 10 amino acid according to the sequent synthesis dengue fever virus NS1 albumen of 2 type Hainan of dengue fever virus strain
8mer-1 (1040-1048, AGPWHLGKL) polypeptide of row；69 polypeptides of acquisition are dissolved into the solution of 100mg/ml with DMSO,
These polypeptides are prepared into 4 kinds of continuous peptide libraries containing 15-18 kind polypeptides, the final concentration of 5.5-6.6mg/ of each peptide library
ml.Polypeptide packing is stored in -20 DEG C, uses a concentration of 2ug/ml.
2nd, purifying protein is immunized mouse and prepares polyclonal antibody
1. all zooperies all formulate to obtain mark according to the nursing of the experimental animal of guest's Western method Leah university and the practical committee
Quasi- guidelines；C57BL male mices (6-8 weeks big) are purchased from River Laboratories.
3. mouse is divided into experimental group and control group, every group each 6, experimental group injects recombinant protein of the present invention, control
The physiological saline of group injection isodose, passes through injection 1010The PBS solution of a adenovirus vector is to the right tibialis anterior of mouse (2 positions
Point, each 25 μ l of site), recombinant adenoviral vector H5-NS1 is immunized.
4. dissecting the mouse after being inoculated with 14 days, the NS1 specific T-cells of mouse is taken to analyze.
Set about in terms of dengue fever virus t cell immune response, vaccine, successful structure are built using simian adenoviral vector
Dengue fever virus Ad carrier bacterin C9-NS1 have been built, immune Best Times and dosage, screening are determined in BALB/c mouse experiment
Identify that H-2d is restricted, NS1 specific T-cells epitope (Fig. 1,2 and table 1) carries out sun with bioinformatics method
Property T cell antigen epitope sequences guard analysis (Fig. 3), it was demonstrated that the epitope have preferable type between cross reaction.For mirror
The t cell epitope made under suitable immunization time and dosage is collected immune mouse spleen lymphocyte progress T cell and is exempted from respectively
The phenotypic analysis of epidemic disease reaction, cytokine secretion functional analysis, CTL functional analyses (Fig. 4).It is small that structure shows that the vaccine can induce
Mouse generates strong CD8+T cell illustrates there is certain immunological memory effect, while CTL experiment in vivo room confirms to generate and kill
Wound property T cell effect.
Table 1 screens the CD8 of DEN2-NS1 albumen on Balb/C mouse+T cell epitope antigen
It should be noted that it should be noted that the experimental method concrete operations that the present invention is previously mentioned are not specifically stated
, it is operated according to ordinary skill in the art means, such as the recombination of NS1 gene chemical synthesis, adenovirus vector and 293 cells
The operating methods such as transfection.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution recorded in foregoing embodiments either to which part or all technical features into
Row equivalent replacement；And these modifications or replacement, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme should all cover in the claim of the present invention and the range of specification.
Sequence table
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<120>Dengue fever virus vaccine
<141> 2018-01-15
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<213>Artificial sequence (artificial sequence)
<400> 1
atggatccaa acactgtgtc aagctttcag gtagattgct ttctttggca tgtccgcaaa 60
cgagttgcag accaagaact aggtgatgcc ccattccttg atcggcttcg ccgagatcag 120
aaatccctaa gaggaagggg cagcactctt ggtctggaca tcgagacagc cacacgtgct 180
ggaaagcaga tagtggagcg gattctgaaa gaagaatccg atgaggcact taaaatgacc 240
atggcctctg tacctgcgtc gcgttaccta accgacatga ctcttgagga aatgtcaagg 300
gaatggtcca tgctcatacc caagcagaaa gtggcaggcc ctctttgtat cagaatggac 360
caggcgatca tggataaaaa catcatactg aaagcgaact tcagtgtgat ttttgaccgg 420
ctggagactc taatattgct aagggctttc accgaagagg gagcaattgt tggcgaaatt 480
tcaccattgc cttctcttcc aggacatact gctgaggatg tcaaaaatgc agttggagtc 540
ctcatcggag gacttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga 600
ttcgcttgga gaagcagtaa tgagaatggg agacctccac tcactccaaa acagaaacga 660
gaaatggcgg gaacaattag gtcagaagtt tgaagaaata a 701
Claims (7)
1. a kind of dengue fever virus vaccine, which is characterized in that the vaccine is genetic vaccine, with E1 gene orders missing
Adenovirus vector H5 is vector construction, and contains dengue fever virus NS1 protein gene.
2. dengue fever virus vaccine as described in claim 1, which is characterized in that further include immunologic adjuvant.
3. dengue fever virus vaccine as claimed in claim 2, which is characterized in that the immunologic adjuvant for Freund's complete adjuvant or
Incomplete Freund's adjuvant.
4. dengue fever virus vaccine as described in claim 1, which is characterized in that the dengue fever virus NS1 protein gene is
The NS1 protein gene sequences of 2 type virus Hainan of dengue fever strain, sequence is as shown in SEQ NO.1.
5. adenovirus vector-NS1 recombinant proteins, which is characterized in that using the adenovirus vector H5 of E1 gene orders missing as carrier,
Acquisition is expressed after importing dengue fever virus NS1 protein gene.
6. a kind of polyclonal antibody, which is characterized in that recombinant protein is as antigen as described in claim 5, through immune response system
It is standby to obtain.
7. one group of immunogenic polypeptide, which is characterized in that synthesized by the NS1 protein gene sequences of 2 type Hainan of dengue fever virus strain
The polypeptide containing 10 amino acid repetitive sequences, it is AGPWHLGKL to repeat peptide sequence.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810034931.1A CN108159410A (en) | 2018-01-15 | 2018-01-15 | Dengue fever virus vaccine |
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| CN201810034931.1A CN108159410A (en) | 2018-01-15 | 2018-01-15 | Dengue fever virus vaccine |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004076664A3 (en) * | 2003-02-21 | 2005-03-24 | Univ South Florida | Vectors for regulating gene expression |
| US8414884B2 (en) * | 2007-09-12 | 2013-04-09 | Aeras Global Tb Vaccine Foundation | Methods to increase transgene expression from bacterial-based delivery systems by co-expressing suppressors of the eukaryotic type I interferon response |
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2018
- 2018-01-15 CN CN201810034931.1A patent/CN108159410A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004076664A3 (en) * | 2003-02-21 | 2005-03-24 | Univ South Florida | Vectors for regulating gene expression |
| US8414884B2 (en) * | 2007-09-12 | 2013-04-09 | Aeras Global Tb Vaccine Foundation | Methods to increase transgene expression from bacterial-based delivery systems by co-expressing suppressors of the eukaryotic type I interferon response |
Non-Patent Citations (3)
| Title |
|---|
| GUANGPING GAO等: ""Adenovirus-Based Vaccines Generate Cytotoxic T Lymphocytes to Epitopes of NS1 from Dengue Virus That Are Present in All Major Serotypes"", 《HUMAN GENE THERAPY》 * |
| HALL,R.M等: ""Influenza A virus (A/Puerto Rico/8/1934(H1N1)) segment 8, complete sequence"", 《GENBANK》 * |
| JIANG,L等: ""Dengue virus type 2 strain Hainan nonstructural glycoprotein NS-1 (NS1) gene, partial cds"", 《GENBANK》 * |
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Application publication date: 20180615 |