CN1966083B - Tumor vaccine of fusion gene and construction method thereof - Google Patents
Tumor vaccine of fusion gene and construction method thereof Download PDFInfo
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Abstract
The invention relates to a fusion gene cancer vaccine and relative production method, wherein it comprises that: recombining DNA to connect bacillus coli thermal-instable toxin B sub gene and cancer antigen gene, to prepare one fusion gene cancer vaccine. The bacillus coli thermal-instable toxin B sub gene is EtxB. The inventive method can generate strong immunity answer reaction on bacterial antigen, and the activated CD4+Th cell in reaction can activate the body fluid immunity reaction and cell immunity reaction on cancer antigen. The technology for fusion gene cancer vaccine provided by the invention is an effective means for overcoming immune system tumour antigen immune tolerance, and is suitable for efficiently enhancing efficacy of tumor vaccine.
Description
Technical field
The invention belongs to gene engineering technology field, relate in particular to tumor vaccine of fusion gene and construction method.
Technical background
At present, at tumor treatment, what extensively adopt is operation, chemotherapy and radiotherapy, though can remove most of tumor cell and most cancer patients' the state of an illness is eased with conventional first-line treatments such as operation, chemotherapy and radiotherapies, but these Therapeutic Method are difficult to reach the effect of eradicating cancerous cell fully, and therefore the recurrence of tumor is inevitable in most patient.An important mechanisms as body defence disease, immune system has the characteristics of high efficiency, high specific and persistency (immunological memory), therefore, utilize the means of active immunity to excite patient's autoimmunity to treat tumor and have very big clinical application potential.
Immune system is made up of innate immune system and adaptive immune system two large divisions, and immunne response is a complexity and the process that is subjected to accurate regulation and control.Pathogen such as virus, antibacterial are structurally more has their peculiar and conservative relatively molecular pattern (Pathogen-Associated Molecular Patterns, be called for short PAMP), when infecting generation, pathogen by the expressed corresponding receptor of PAMP and host cell effect and at first activate innate immune system, show as generation, the release of inflammatory cytokine, chemotactic factor etc.These factors not only have pathogen directly kills and wounds and inhibitory action, but also the DC that attraction and activation play a crucial role in antigen presentation.Subsequently, be activated and the DC of capture antigen migrates to around the lymph node etc. lymphatic organ and activates adaptive immune system at this, show as the B cell with high degree of specificity and memory characteristic and the activation of T cell.The close fit of this innate immune system and acquired immune system had both guaranteed that body can produce reaction and persistent monitoring rapidly to the infringement of external pathogen, also avoided exciting to produce immunoreactive danger from body simultaneously.In these a series of processes, DC has constituted key one ring in innate immune system and the organic connection of acquired immune system, CD4
+T helper cell (CD4
+Th) then pass through to discharge the mode of cytokine and direct cells contacting conducted signal to B cell and CD8
+The setting up of activation, expansion and immunological memory of cytotoxic T lymphocyte (Cytotoxic T Lymphocyte is called for short CTL) played very important effect in the process.Most of tumor cells expression tumor associated antigen (Tumor-Associated Antigen, be called for short TAA), and in tumorigenic pathological process, can cause the release of some inflammatory cytokines, heat shock protein owing to reasons such as tissue injury and necrocytosiss.Therefore, infect as treating microbial pathogens, immune system should monitor this " danger " signal that discharges when tumor is grown fast and make corresponding reaction to suppress tumor growth.A large amount of zooperies and clinical data also confirm the immunoreation that immune system really can be spontaneous to making of tumor, and some clinical trials show that further active immunity (vaccine therapy) can obtain certain therapeutic effect in some patient.Yet, compare in the success that is obtained aspect the infection prevention disease with the antimicrobial vaccine, the effectiveness of present most of cancer vaccines also reaches gratifying degree far away, its main cause be these vaccines effectively activating immune system to the immunoreation of " self property " tumor antigen.Therefore, how overcoming immune system is the key issue that can tumor vaccine obtain the required solution of satisfactory effect clinically to the immunologic tolerance of self property tumor antigen.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of and can overcome immune system the toleration of tumor antigen, the tumor vaccine of fusion gene that the enhancing antitumor is renderd a service.
Another object of the present invention provides a kind of construction method of tumor vaccine of fusion gene.
Technical scheme of the present invention is summarized as follows:
A kind of tumor vaccine of fusion gene, make up with the following step: be connected with tumor antigen gene with the method for the recombinant DNA escherichia coli thermal instability toxin B subunit gene of will encoding, promptly make a kind of tumor vaccine of fusion gene, described escherichia coli thermal instability toxin B subunit gene is called for short EtxB.
The method of attachment of described EtxB and tumor antigen gene is: with sequence table SEQ ID No.1, SEQ ID No.2 is the PCR primer, with the described EtxB DNA sequence of SEQ ID No.3 is template, amplification, the EtxB fragment that obtains NotI and XbaI digestion with restriction enzyme, obtain the 320bp dna fragmentation, with pcDNA3 vector plasmid with NotI and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pEtxB, again with sequence table SEQ IDNo.4, SEQ ID No.5 is the PCR primer, with the described BCL1 scFv of SEQ ID No.6 DNA sequence is template, amplification, the BCL1 scFv fragment that obtains HindIII and NotI digestion with restriction enzyme, obtain the 796bp dna fragmentation, described pEtxB vector plasmid with HindIII and NotI digestion with restriction enzyme carries out coupled reaction with the T4 dna ligase, makes pBCL1-EtxB.
The method of attachment of described EtxB and tumor antigen gene is: with sequence table SEQ ID No.7, SEQ ID No.8, SEQID No.9, SEQ ID No.10 and SEQ ID No.2 are the PCR primer, merge with will the encode dna fragmentation of tPA signal peptide and described EtxB of the method for PCR, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain 429 bp dna fragmentations, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make ptPA-EtxB, the method for reuse PCR is a template with described ptPA-EtxB plasmid DNA, SEQ ID No.7 and SEQ ID No.11 are that primer is coding CT26 tumor CD8
+The dna fragmentation AH1 of cytotoxic T lymphocyte epitope and tPA-EtxB merge, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain the 456bp dna fragmentation, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pEtxB-AH1.
A kind of tumor vaccine of fusion gene construction method, form by following step: be connected with tumor antigen gene with the method for the recombinant DNA escherichia coli thermal instability toxin B subunit gene of will encoding, promptly make a kind of tumor vaccine of fusion gene, described escherichia coli thermal instability toxin B subunit gene is called for short EtxB.
The method of attachment of described EtxB and tumor antigen gene is: with sequence table SEQ ID No.1, SEQ ID No.2 is the PCR primer, with the described EtxB DNA sequence of SEQ ID No.3 is template, amplification, the EtxB fragment that obtains NotI and XbaI digestion with restriction enzyme, obtain the 320bp dna fragmentation, with pcDNA3 vector plasmid with NotI and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pEtxB, again with sequence table SEQ IDNo.4, SEQ ID No.5 is the PCR primer, with the described BCL1 scFv of SEQ ID No.6 DNA sequence is template, amplification, the BCL1 scFv fragment that obtains HindIII and NotI digestion with restriction enzyme, obtain the 796bp dna fragmentation, described pEtxB vector plasmid with HindIII and NotI digestion with restriction enzyme carries out coupled reaction with the T4 dna ligase, makes pBCL1-EtxB.
The method of attachment of described EtxB and tumor antigen gene is: with sequence table SEQ ID No.7, SEQ ID No.8, SEQID No.9, SEQ ID No.10 and SEQ ID No.2 are the PCR primer, merge with will the encode dna fragmentation of tPA signal peptide and described EtxB of the method for PCR, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain the 429bp dna fragmentation, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make ptPA-EtxB, the method for reuse PCR is a template with described ptPA-EtxB plasmid DNA, SEQ ID No.7 and SEQ ID No.11 are that primer is coding CT26 tumor CD8
+The dna fragmentation AH1 of cytotoxic T lymphocyte epitope and tPA-EtxB merge, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain the 456bp dna fragmentation, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pEtxB-AH1.
The present invention adopts the dna vaccination design that weak immunogenic tumor antigen and strong immunogenic bacterial antigens (EtxB) are connect mutually.Will being intended to immune system and can producing very strong immune response of this fusion bacterin design, and the antigenic CD4 of the directed toward bacteria that in reaction, is activated to bacterial antigens
+The Th cell can activate humoral immune reaction and the cell immune response at tumor antigen by the immunology principle of link auxiliary (Linked Help), a kind of tumor vaccine of fusion gene technology of the present invention is to overcome the effective means of immune system to the tumor antigen immunologic tolerance, is applicable to the effectiveness of efficient enhancing tumor vaccine.
Description of drawings
Fig. 1 is the construction strategy of vector plasmid pEtxB.
Fig. 2 is the construction strategy of tumor vaccine of fusion gene pBCL1-EtxB.
Fig. 3 is the construction strategy of vector plasmid ptPA-EtxB.
Fig. 4 is the construction strategy of tumor vaccine of fusion gene pEtxB-AH1.
Fig. 5. tumor vaccine of fusion gene pBCL1-EtxB energy inducing mouse produces the survival rate behind anti-BCL1 immune globulin antibody and the significant prolongation mouse inoculation tumor cell.
Fig. 6. tumor vaccine of fusion gene pEtxB-AH1 energy inducing mouse produces the survival rate behind reaction of AH1 specific T-cells and the significant prolongation mouse inoculation tumor cell.
The specific embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1: the structure of tumor vaccine of fusion gene pBCL1-EtxB
With sequence table SEQ ID No.1, SEQ ID No.2 is the PCR primer, with the described EtxB DNA sequence of SEQ ID No.3 is template, amplification is also introduced NotI and XbaI enzyme cutting site, the volume of PCR reaction is that 50 μ l contain: the described EtxB DNA of 0.1 μ gSEQ IDNo.3 masterplate, 20pmol SEQ ID No.1 and SEQ ID No.2,5 μ l 250mMol dNTPs, 2.5U Taq archaeal dna polymerase (German Qiagen company) and 5 μ l, 10 * PCR buffer, the PCR reaction condition is: 94 ℃ of 5min.; 94 ℃ of 30sec., 50 ℃ of 30sec., 72 ℃ of 45sec., circulation 5 is taken turns; 94 ℃ of 30sec., 60 ℃ of 30sec., 72 ℃ of 45sec., EtxB the fragment NotI and the XbaI digestion with restriction enzyme of acquisition taken turns in circulation 25, obtain the 320bp dna fragmentation, pcDNA3 (Invitrogen company) vector plasmid with NotI and XbaI digestion with restriction enzyme carries out coupled reaction with the T4 dna ligase, makes pEtxB, construction strategy is seen Fig. 1, (ID 1 is SEQ ID No.1 among the figure, and ID 2 is SEQ ID No.2, other by that analogy);
Be the PCR primer with sequence table SEQ ID No.4, SEQ ID No.5 again, with the described BCL1 scFvDNA of SEQ ID No.6 sequence is template, HindIII and NotI restriction enzyme site are introduced in amplification, the volume of PCR reaction is that 50 μ l contain: 0.1 μ gBCL1 scFv DNA masterplate, 20pmol SEQ ID No.4 and SEQ ID No.5 PCR primer, 5 μ l 250mMol dNTPs, 2.5U Taq archaeal dna polymerase and 5 μ l, 10 * PCR buffer, the PCR reaction condition is: 94 ℃ of 5min.; 94 ℃ of 30sec., 50 ℃ of 30sec., 72 ℃ of 45sec., circulation 5 is taken turns; 94 ℃ of 30sec., 60 ℃ of 30sec., 72 ℃ of 45sec., circulation 25 is taken turns, and the BCL1 scFv fragment of acquisition obtains 796 bp dna fragmentations with HindIII and NotI digestion with restriction enzyme, with described pEtxB vector plasmid with HindIII and NotI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pBCL1-EtxB, construction strategy is seen Fig. 2.
Embodiment 2: the structure of tumor vaccine of fusion gene pEtxB-AH1
With sequence table SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10 and ID No.2 are the PCR primer, merge with will the encode dna fragmentation of tPA signal peptide and described EtxB of the method for PCR, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain the 429bp dna fragmentation, the volume of PCR reaction is that 50 μ l contain: 0.1 μ g EtxB DNA masterplate, 20pmol SEQ ID No.7,2pmol SEQ ID No.8,2pmol SEQ ID No.9,2pmol SEQ ID No.10 and 20pmol SEQ ID No.2 primer, 5 μ l 250mMol dNTPs, 2.5U Taq archaeal dna polymerase and 5 μ l, 10 * PCR buffer, the PCR reaction condition is: 94 ℃ of 5min.; 94 ℃ of 30sec., 50 ℃ of 30sec., 72 ℃ of 45sec., circulation 5 is taken turns; 94 ℃ of 30sec., 60 ℃ of 30sec., 72 ℃ of 45sec., circulation 25 is taken turns, the EtxB fragment that amplification obtains HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the pcDNA3 vector plasmid (Invitrogen company) with the digestion of same restrictions restriction endonuclease with the T4 dna ligase, link acquisition ptPA-EtxB, construction strategy is seen Fig. 3;
The method of reuse PCR is that template, SEQ ID No.7 and SEQ ID No.11 are that primer is coding CT26 tumor CD8 with the ptPA-EtxB plasmid DNA
+The dna fragmentation AH1 of cytotoxic T lymphocyte epitope and tPA-EtxB merge, the volume of PCR reaction is that 50 μ l contain: 0.1 μ g ptPA-EtxB DNA, 20pmol SEQ ID No.7 and SEQ IDNo.11PCR primer, 5 μ l 250mMol dNTPs, 2.5U Taq archaeal dna polymerase and 5 μ l, 10 * PCR buffer, the PCR reaction condition is: 94 ℃ of 5min.; 94 ℃ of 30sec., 50 ℃ of 30sec., 72 ℃ of 45sec., circulation 5 is taken turns; 94 ℃ of 30sec., 60 ℃ of 30sec., 72 ℃ of 45sec., circulation 25 is taken turns, and the PCR product that is obtained obtains the 456bp dna fragmentation through HindIII and XbaI digestion with restriction enzyme, with pcDNA3 vector plasmid (Invitrogen company) with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4 dna ligase, make pEtxB-AH1, construction strategy is seen Fig. 4.
The factor that influences the cancer vaccine effectiveness is a lot, and how breaking immune system then is a most key link to the toleration of tumor antigen.This is because most of TAA are antigen rather than the exogenous antigens that come from self, so can discern the quantity of T cell (being called for short the TAA-T cell) of the TAA of this oneself's property and they in the integral body of T cell constitutes all is very limited to the affinity of TAA, this at first is because those have than the T cell of affinity by force TAA and just have been removed in thymus under the regulation and control of central immunologic tolerance mechanism.In addition, those TAA-T cells of not removed by central immunologic tolerance mechanism because affinity is lower also can be under the effects of periphery immunologic tolerance mechanism after entering peripheral-system or are eliminated or lose respond to TAA.Equally, the integral body of B cell constitutes the regulation and control that also are subjected to similar immunologic tolerance mechanism.Because CD4
+Th is at B cell and CD8
+The decisive role in the process is set up in CTL immunoreation (activate, expand and memory), how to overcome CD4
+The Th system is the key issue that institute must solution in the tumor vaccine research to the toleration of tumor antigen.To this, we have adopted tumor antigen and the vaccine design with strong immunogenic escherichia coli thermal instability toxin B subunit (EtxB) fusion.The immune system that will be intended to of this fusion bacterin does not have toleration to this exogenous antigen of EtxB, therefore can produce very strong immune response to it, and the antigenic CD4 of the directed toward bacteria that in reaction, is activated
+The Th cell can be by efficient humoral immune reaction and the cell immune response that activates at tumor antigen of the immunology principle of link auxiliary (Linked help).In addition, the EtxB decapacitation is played to be provided outside the auxiliary effect of link, and itself also has the effect that directly activates T, B and DC cell.Also have, different with most proteantigens, EtxB can enter cell and activate CD8 by MHC-I (Major Histocompatibility Class I) approach in receptor-mediated endocytosis mode
+Ctl response, this is to activating CD8 by intersection submission mechanism (Cross Priming)
+CTL has important facilitation.
Material
1) bacterial strain and cell line
E. coli host bacteria JM103 is from Promega company.
BCL1 is mouse B cell leukemia/lymphocytic cancer cell system and expresses IgM/ λ, and 293T cell line is the human embryo kidney epithelial cell, and CT26 is a mice colon-cancer cell system, more than three kinds of cell lines all from ATCC cell preservation center.
2) EtxB gene and BCL1 scFv gene
The EtxB gene is according to the nucleotide sequence chemosynthesis of document 1 report.Nucleotide sequence and document 3 reported method that BCL1 scFv genetic fragment is pressed document 2 reports make up.
3) genetic engineering toolenzyme and other chemical reagent
Restriction restriction endonuclease, T4 dna ligase, Taq archaeal dna polymerase and molecular biology and required chemicals of cell culture and culture medium etc. are available from companies such as Promega, Invitrogen, New England Biolabs and Sigma.
Method
Except that the experimental technique of following detailed description, molecular cloning and other DNA genetic manipulation are the laboratory routine techniques, referring to Sambrook J, and Fristsh EF, Maniatis T.Molecular Cloning:A Laboratory Manual 2
NdEd.NY, Cold Spring Harbor Laboratory Press, 1989.
Embodiment 3: the research of tumor vaccine of fusion gene pBCL1-EtxB inducing antitumor immunity reaction
ELISA detects the antibody response of mice to the pBCL1-EtxB anti-tumor vaccine: select the 4-6 BALB/c mouse in age in week for use, each 8 of pBCL1-EtxB experimental group, pBCL1, pEtxB vehicle Control group and normal saline negative control group.With physiological saline solution the dna vaccination plasmid vector is dissolved to 0.5mg/ml, injects 50 μ l dna vaccinations respectively at two hind leg musculus quadriceps places of mice then.Later on every in kind carrying out twice totally booster immunization injection 3 weeks.Before each vaccination, getting blood, be used for the ELISA antibody test through blood coagulation, centrifugal back acquisition serum from mouse tail.The result shows that the mice of accepting pBCL1-EtxB and pEtxB immunity inoculation has all produced anti-EtxB antibody response, the then not anti-EtxB antibody response of pBCL1 and normal saline experimental group.The mice of accepting the pBCL1-EtxB inoculation has produced anti-BCL1 IgM antibody response, accepts then nonreactive BCL1 IgM antibody response of pEtxB and pBCL1 inoculation and normal saline mice in control group.
The tumor inoculation experiment detects the protective effect of pBCL1-EtxB anti-tumor vaccine to mice: after the 3rd 3 weeks of inoculation, import 2.5 * 10 by intravenous injection to mice
5After this BCL1 tumor cell observes the growing state of tumor in mice every day.Observe to find, have only the pBCL1-EtxB inoculation could suppress effectively the BCL1 tumor in the intravital growth of mice, significantly improve the survival rate (Fig. 5) of mice.
Above result shows that the dna vaccination of only expressing BCL1scFv can not effectively activate the B cell effect, but pBCL1-EtxB fusion bacterin inducing antitumor B cell immune response very effectively then.
Embodiment 4: the research of tumor vaccine of fusion gene pEtxB-AH1 inducing antitumor immunity reaction
ELISA detects the antibody response of mice to the pEtxB-AH1 anti-tumor vaccine: select the 4-6 BALB/c mouse in age in week for use, each 8 of pEtxB-AH1 experimental group, pEtxB empty carrier matched group and normal saline negative control group.With physiological saline solution the dna vaccination plasmid vector is dissolved to 0.5mg/ml, injects 50 μ l dna vaccinations respectively at two hind leg musculus quadriceps places of mice then.Later on every in kind carrying out 1 booster immunization injection 3 weeks.Before each vaccination, getting blood, be used for the ELISA antibody test through blood coagulation, centrifugal back acquisition serum from mouse tail.The result shows that the mice of accepting pEtxB-AH1 and pEtxB immunity inoculation has all produced anti-EtxB antibody response, but the then not anti-EtxB antibody response of normal saline experimental group.
The tumor inoculation experiment detects the protective effect of pEtxB-AH1 anti-tumor vaccine to mice: after the 2nd 3 weeks of inoculation, import 1 * 10 by intravenous injection to mice
5After this CT26 tumor cell observes the growing state of tumor in mice every day.Observe to find, have only the pBCL1-EtxB inoculation could suppress effectively the CT26 tumor in the intravital growth of mice, significantly improve the survival rate (Fig. 6) of mice.
Sequence table
＜110〉Tianjin Agsai Cell Gene Engineering Co., Ltd
＜120〉tumor vaccine of fusion gene and construction method
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<211>31
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<211>30
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＜213〉artificial sequence
<221>misc_difference
<222>(1)…(30)
<400>2
tatatctaga?ctagttttcc?atactgattg?30
<210>3
<211>312
<212>DNA
＜213〉artificial sequence
<221>misc_difference
<222>(1)…(312)
<400>3
gctcctcagtctattacagaactatgttcggaatatcacaacacacaaatatatacgata
aatgacaagatactatcatatacggaatcgatggcaggcaaaagagaaatggttatcatt
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<221>misc_difference
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＜213〉artificial sequence (fraction is an artificial sequence, and is most of from mice (Mus musculus))
<221>misc_difference
<222>(1)…(783)
<400>6
atgggttggagctgtatcatcttctttctggtagcaacagctacaggtgtgcactcccag
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tcaggcggaggtggctctggcggtggcggatcgcaggctgttgtgactcaggaatctgca
ctcaccacatcacctggtgaaacagtcacactcacttgtcgctcaagtactggggctgtt
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ggagacaaggctgccctcaccatcacaggggcacagactgaggatgaggcaatatatttc
tgtgctctatggtacagcaaccattttattttcggcagtggaaccaaggtcactgtccta
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＜213〉artificial sequence
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tataaagcttgccaccatggatgcaatgaagagagggctctgctgtgtg
<210>8
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<212>DNA
＜213〉artificial sequence
<221>misc_difference
<222>(1)…(48)
<400>8
gagggctctgctgtgtgctgctgctgtgtggagcagtcttcgtttcgc
<210>9
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<2I2>DNA
＜213〉artificial sequence
<221>misc_diffrence
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<400>9
gagcagtcttcgtttcgcccagccaggaaatccatgcccgattcagaag
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<211>50
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＜213〉artificial sequence
<221>misc_difference
<222>(1)…(50)
<400>10
tccatgcccgattcagaagaggcgccagatctgctcctcagtctattaca
<210>11
<211>58
<212>DNA
＜213〉artificial sequence
<221>misc_difference
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Claims (1)
1. tumor vaccine of fusion gene construction method, it is characterized in that forming: be connected with tumor antigen gene with the method for the recombinant DNA escherichia coli thermal instability toxin B subunit gene of will encoding by following step, promptly make a kind of tumor vaccine of fusion gene, described escherichia coli thermal instability toxin B subunit gene is called for short EtxB, the method of attachment of described EtxB and tumor antigen gene is: with sequence table SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10 and SEQ ID No.2 are the PCR primer, merge with will the encode dna fragmentation of tPA signal peptide and the described EtxB of SEQ ID No.3 of the method for PCR, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain dna fragmentation, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4DNA ligase, make ptPA-EtxB, the method for reuse PCR is a template with described ptPA-EtxB plasmid DNA, SEQ ID No.7 and SEQ ID No.11 are that primer is coding CT26 tumor CD8
+The dna fragmentation AH1 of cytotoxic T lymphocyte epitope and tPA-EtxB merge, the PCR product that is obtained is through HindIII and XbaI digestion with restriction enzyme, obtain dna fragmentation, with pcDNA3 vector plasmid with HindIII and XbaI digestion with restriction enzyme, carry out coupled reaction with the T4DNA ligase, make pEtxB-AH1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1561232A (en) * | 2000-12-11 | 2005-01-05 | 布里斯托尔大学 | Therapeutic agent comprising a B-subunit of a protein toxin |
| CN1853731A (en) * | 2005-04-26 | 2006-11-01 | 中国人民解放军军事医学科学院生物工程研究所 | Use of staphylococcal enterotoxin A gene and its coded protein |
-
2006
- 2006-11-27 CN CN2006101295994A patent/CN1966083B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1561232A (en) * | 2000-12-11 | 2005-01-05 | 布里斯托尔大学 | Therapeutic agent comprising a B-subunit of a protein toxin |
| CN1853731A (en) * | 2005-04-26 | 2006-11-01 | 中国人民解放军军事医学科学院生物工程研究所 | Use of staphylococcal enterotoxin A gene and its coded protein |
Non-Patent Citations (4)
| Title |
|---|
| Haynes JR et al..Potent protective cellular immune responses generated by a DNA vaccine encoding HSV-2 ICP27 and the E. coli heat labile enterotoxin.《Vaccine》.2006,第24卷(第23期),5016–5026. * |
| Leong J et al..E.coli (from human) heat-labile enterotoxin subunit B gene (eltB), complete cds,M17874,588 bp,DNA linear.《NCBI》.1996,1-2. * |
| LeongJetal..E.coli(fromhuman)heat-labileenterotoxinsubunitBgene(eltB) complete cds，M17874，588 bp，DNA linear.《NCBI》.1996 |
| wang J et al..Enhanced immunization after intranasal coadministration of Escherichia coli heat-labile enterotoxin B subunit and human papillomavirus 16-L1 DNA vaccine.《Chin Med J》.2006,第119卷(第5期),408-411. * |
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