CN109456945A - A kind of application for improving DC cell HPV viruse antigen and offering DC cell after the gene modification method and gene modification of efficiency - Google Patents
A kind of application for improving DC cell HPV viruse antigen and offering DC cell after the gene modification method and gene modification of efficiency Download PDFInfo
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Abstract
The present invention provides the gene modification method and its application that a kind of raising DC cell HPV viruse antigen offers efficiency, are related to gene modification technical field.The method of the invention, comprising: building pGEM-T-rHPV16-E6E7 plasmid makes it realize prokaryotic expression in host strain；The in-vitro transcription process of T7 promoter driving, obtains the HPV16 mRNA molecule of improvement；Molecule transfects DC cell, obtains the DC cell of gene modification.The DC cell through gene modification obtained, the expression of MHC-I class and II class epitope in DC cell can be significantly increased, antigen submission is carried out by MHC-I classpath, generate CD4+ and CD8+ killer T cell, repair patient-specific humoral immunity and cellular immunity, and then HPV16 infected tissue cell is killed, the HPV16 virus of Long-term Infection is removed comprehensively.
Description
Technical field
The invention belongs to gene modification technical fields, and in particular to a kind of raising DC cell HPV viruse antigen offers efficiency
Gene modification method and gene modification after DC cell application.
Background technique
Dendritic Cells (DC cell) derives from marrow CD34+Candidate stem cell because the pseudopodium of protrusion is as branch, therefore is named
For Dendritic Cells, Lymphatic System DC and medullary system DC two major classes, the precursor and NK cell, T of Lymphatic System DC are divided into according to source difference
Cell is identical, and medullary system DC precursor is identical as monocyte and granulocyte.It is variant according to maturity difference DC function, not at
Ripe DC includes important organelle, including inner body or MHClI class cell and lysosome etc., and height expresses complement, Toll-like receptor,
Antigen is absorbed for DC, and structure basis is provided；Maturation is then divided into after immature DC absorbs antigen or by the stimulation of certain factors
DC, height expression costimulating factor (CD80, CD86, CD40, CD40L etc.) and adhesion factor (IL12p35, IL12p40,
IL12p70, IFN-γ etc.), obtain the ability of present antigen (MHC-I and MHC-II) and activating T cell.
2002, Duma etc. was proposed " swollen on the basis of summarizing forefathers' research by a large amount of bases and clinical test verifying
Tumor immunoediting " is assumed, it is believed that tumour, which is formed in immune response, is divided into 3 stages: removing, balance, escape, wherein DC is exempting from
It plays a key effect in epidemic disease response.In the occurrence and development of tumour, after canceration occurs for host cell, immature DC is through chemotactic
Effect enters tumor microenvironment from peripheral blood, forms maturation DC under tumour antigen stimulation, raises antigen submission ability, height expression
Costimulating factor, intake tumour antigen are combined into MHC- antigenic peptide complexes as the first activation signals with MHC and activate initial T thin
Born of the same parents, which are allowed to activation differentiation, becomes t helper cell (Th) and Cytotoxic T lymphocytes (CTL), disappears to start humoral immune response
It goes out and expresses the cancerous tumor cell of HLA polypeptide；High expression IL-12, IL-6, TNF-a and IL-10 simultaneously, enhance natural killer cells
Direct killing effect, as the second activation signals enhancing body to the immune response of inflammation or tumour.The chemotactic factor (CF) of DC secretion
Can specificity chemotactic primary tape T cell, promote T cell in the aggregation of tumor locus, enhancing T cell excitation is corresponding immune to answer
It answers.However, DC identification and intake can be escaped due to the unstability and heterogeneity of tumour cell heredity, it is thin so as to cause tumour
Born of the same parents, which are constantly proliferated, even to be shifted.
Have research to confirm: the general low expression of tumour cell does not express MHC I, MHC class Ⅱmolecule and cell adhesion molecule,
VEGF is discharged simultaneously, and TGF-β hinders DC differentiation and maturation, is unfavorable for processing and forms MHC- antigenic peptide complexes, to lose antigen
Function is presented, tumor proliferation and transfer finally occurs.Also isolated from tumour studies have shown that: compared with Normal donor DC
DC obviously lack antigen presentation function, further support the hypothesis.And current DC vaccine can induce specificity carefully
Cytotoxic T cells (CTL) react and infection are promoted to subside, but there are operating procedure complexity, the operating time is long, and DC cell is tieed up
Hold antigen offer the time it is short the disadvantages of.
Summary of the invention
In view of this, the purpose of the present invention is to provide the genes that a kind of raising DC cell HPV viruse antigen offers efficiency
The application of DC cell after method of modifying and gene modification, the DC vaccine of the gene modification can express holoprotein sequence, and wrap
Containing all antigen sites；DC cellular antigens submission can be enhanced with long-term expression antigen and cell factor or costimulatory molecules
Ability, promote the activation of T cell.May also help in inhibition of the DC cells against neoplastic to it simultaneously, extend shelf-life and
Promote the activation of T cell, NK cell.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of raising DC cell HPV viruse antigens to offer the gene modification method of efficiency, including following
Step: 1) Fusion gene construction into pGEM-T vector plasmid is obtained into recombinant plasmid pGEM-T-rHPV16-E6E7；It is described to melt
The sequence of gene is closed as shown in SEQ ID NO.1；
2) the recombinant plasmid pGEM-T-rHPV16-E6E7 is linearized through restriction enzyme SpeI, to obtain
Linearization plasmid as template, start the in-vitro transcription process of T7 promoter driving, obtain the HPV16 mRNA molecule of improvement；
3) DC cell is transfected using the HPV16 mRNA molecule of the improvement, obtains the DC cell of gene modification.
It preferably, successively include: E6, E7 of Kozak sequence, signal peptide, HPV16 hypotype in the step 1) fusion
Sequence and molecular transport signal.
Preferably, the system of the step 2) in-vitro transcription is 20 μ L systems, comprising:
It preferably, further include the HPV16 mRNA molecule that the improvement is concentrated before the step 3) transfection, the concentration
Method includes: that 30 μ L nuclease free distilled waters and 30 μ L lithium chloride precipitated liquids are added into the reaction system of in-vitro transcription, and -20 DEG C
Place 30~60min；Under the conditions of 4 DEG C, centrifugation removal supernatant collects RNA precipitate；Using RNA precipitate described in TE buffer solution,
It carries out quantitative to RNA solution with ultraviolet specrophotometer and is saved in -70 DEG C.
Preferably, the lithium chloride precipitated liquid includes the component of following content: the EDTA of the LiCl and 50mM of 7.5M, described
The pH value of lithium chloride precipitated liquid is 8.0.
Preferably, the method for the step 3) transfection is electrotransfection.
Preferably, the cultural method of step 3) the DC cell, comprising: the peripheric venous blood 50mL after utilizing infection HPV,
Add the centrifugation layering of 25mL lymphocyte separation medium, takes the cell of second layer mononuclearcell layer, trained using serum-free X-VIVO-15
It supports base to be diluted the cell, in 37 DEG C of 5%CO2It is incubated for 2.5 hours in incubator, obtains adherent mononuclearcell
Layer；Suspension cell is washed away with PBS, is added white containing 800IU/mL granulocyte/macrophage colony stimulating factor and 500IU/mL
The X-VIVO-15 culture medium culture of -4 cell factor of interleukin 24 hours, obtains immature Dendritic Cells；Every 3 days half amounts are changed
Liquid is primary, and supplies granulocyte/macrophage colony stimulating factor and interleukin-4 cell factor；In X-VIVO-15 culture medium
Recombination humanTNF-α, IL-1b, IL-6 and PGE2 is added, induces DC cell maturation.
Preferably, the infection HPV is infection HPV16 hypotype and development is CIN2 and CIN3 period.
Preferably, in the X-VIVO-15 culture medium, the final concentration of 10ng/mL of the TNF, the end of the IL-1b
Concentration is 10ng/mL, the final concentration of 1mg/mL of the final concentration of 1000U/mL of the IL-6, the PGE2.
It is special in preparation induction that the present invention also provides the DC cells of the gene modification obtained using said gene method of modifying
Application in specific immunological response drug.
The present invention provides a kind of gene modification methods that raising DC cell HPV viruse antigen offers efficiency, after modification
MRNA sequence carry out DC cell modification, make cell can be with continuous expression antigen, and stimulate DC cell maturation, DC cell can be improved
Specificity offers the ability of HPV16 type viral antigen, and then increases the self immunocyte quantity for killing HPV16 virus, and mention
High immunocyte killing activity enhances immune function, reaches and thoroughly removes HPV16 virus, the purpose for preventing cervical lesions from developing.
Infectious viral particle will not be replicated using the method for the invention, but general immunity can be induced and MHC-I, MHC-II are restricted
Ctl response, and do not had safety good, the strong feature of validity by the interference of internal existing carrier antibody.
Detailed description of the invention
Fig. 1 is fusion structural schematic diagram；
Fig. 2 is the structural schematic diagram and Cytokine Expression Level of fusion protein；
Fig. 3 is CD4 in peripheral blood after being inoculated with DC vaccine of the present invention+/CD8+T cell quantity.
Specific embodiment
The present invention provides a kind of raising DC cell HPV viruse antigens to offer the gene modification method of efficiency, including following
Step: 1) Fusion gene construction into pGEM-T vector plasmid is obtained into recombinant plasmid pGEM-T-rHPV16-E6E7；The fusion
The sequence of gene is as shown in SEQ ID NO.1；
2) the recombinant plasmid pGEM-T-rHPV16-E6E7 is linearized through restriction enzyme SpeI, to obtain
Linearization plasmid as template, start the in-vitro transcription process of T7 promoter driving, obtain the HPV16 mRNA molecule of improvement；
3) DC cell is transfected using the HPV16 mRNA molecule of the improvement, obtains the DC cell of gene modification.
In the gene modification method that raising DC cell HPV viruse antigen of the present invention offers efficiency, by fusion
It constructs into pGEM-T vector plasmid, obtains recombinant plasmid pGEM-T-rHPV16-E6E7；The sequence of the fusion such as SEQ
Shown in ID NO.1.The structural schematic diagram of fusion of the present invention as shown in Figure 1: successively comprising Kozak sequence, signal peptide,
E6 the and E7 sequence and molecular transport signal of HPV16 hypotype, and Kozak sequence is added at 5 ' ends of signal peptide sequence
(gccacc), the translation efficiency of albumen, signal peptide sequence codified transmembrane protein and molecular transport signal are improved.The present invention is to institute
Stating Fusion gene construction, there is no particular determinations into the method for pGEM-T vector plasmid, are preferably directly connected to through T4 ligase into load
Body, the method is as follows:
Following ingredients are added in 0.2mL reaction tube:
Mending ultrapure water to total volume is 10 μ L, can be with 16 DEG C or 4 DEG C overnight.
Conversion:
1. by 50 μ L competent cell JM109 or DH5 α in thawing on ice bath；
2. 5 μ L connection products and competent cell are mixed, ice bath 30min；
3.42 DEG C heat shock 90 seconds, ice bath 3-5min immediately.
After recombinant plasmid pGEM-T-rHPV16-E6E7, the present invention is by the recombinant plasmid pGEM-T-rHPV16-E6E7
It is linearized through restriction enzyme SpeI, the linearization plasmid to obtain starts the body of T7 promoter driving as template
Outer transcription obtains the HPV16 mRNA molecule of improvement.There is no particular determinations for method of the present invention to the linearisation, utilize
Conventional enzymatic cleavage methods of the invention.The system of in-vitro transcription of the present invention is preferably 20 μ L systems, is specifically included:
The method of in-vitro transcription of the present invention preferably carries out in a water bath, and the temperature of the water-bath is preferably 37 DEG C, institute
The time for stating water-bath is preferably 2h.For the present invention before carrying out the water-bath, it is also preferable to include softly blow and beat solution with pipettor to make
It is mixed well, and 2000rpm is centrifuged 30 seconds, is collected liquid in PCR and is reacted bottom of the tube.
After the HPV16 mRNA molecule that must be improved, the present invention is thin using the HPV16 mRNA molecule transfection DC of the improvement
Born of the same parents obtain the DC cell of gene modification.It is also preferable to include the HPV16 mRNA that the improvement is concentrated to divide before the transfection by the present invention
The method of son, the concentration preferably includes: 30 μ L nuclease free distilled waters and 30 μ L being added into the reaction system of in-vitro transcription
Lithium chloride precipitated liquid, -20 DEG C of 30~60min of placement；Under the conditions of 4 DEG C, centrifugation removal supernatant collects RNA precipitate；It is buffered using TE
Liquid dissolves the RNA precipitate, carries out quantitative to RNA solution with ultraviolet specrophotometer and saves in -70 DEG C.Chlorine of the present invention
Change the component that lithium precipitated liquid preferably includes following content: the EDTA of the LiCl and 50mM of 7.5M, the pH of the lithium chloride precipitated liquid
Value is 8.0.For the present invention after the collection RNA precipitate, it is also preferable to include 1mL ethyl alcohol is added into the RNA precipitate that collection obtains
RNA precipitate is washed, and under the conditions of 4 DEG C, RNA precipitate is collected by centrifugation again.Ethyl alcohol of the present invention is preferably ethanol water,
The volume fraction of the ethanol water is preferably 60~80%, and more preferably 65~75%, most preferably 70%.The present invention couple
The method of the transfection is specifically included there is no particular determination, preferably electrotransfection: according to 1g/106The dosage of cell is turned
Dye.MRNA is placed in the electric revolving cup of sterilizing, with Gene Pulser Xcell electroporation complete system (article No. 1652660,
Bio-Rad company) with 300V/150F progress electrotransfection.There is no particular determinations in source of the present invention to the DC cell, preferably
Using homemade DC cell, cultural method preferably includes: to add 25mL to drench using the peripheric venous blood 50mL after infection HPV
The centrifugation layering of bar cell separating liquid, takes the cell of second layer mononuclearcell layer, using serum-free X-VIVO-15 culture medium to institute
It states cell to be diluted, in 37 DEG C of 5%CO2It is incubated for 2.5 hours in incubator, obtains adherent mononuclearcell layer；It is washed with PBS
Suspension cell is removed, is added and contains 800IU/mL granulocyte/macrophage colony stimulating factor and 500IU/mL interleukin-4 cell
The X-VIVO-15 culture medium culture of the factor 24 hours, obtains immature Dendritic Cells；It changes the liquid once in half, and mends within every 3 days
Sufficient granulocyte/macrophage colony stimulating factor and interleukin-4 cell factor；Recombined human is added in X-VIVO-15 culture medium
TNF-α, IL-1b, IL-6 and PGE2 induce DC cell maturation.Infection HPV of the present invention preferably infect HPV16 hypotype and
Development is CIN2 and CIN3 period.In the X-VIVO-15 culture medium, the final concentration of the TNF is preferably 10ng/mL, institute
State the final concentration of 10ng/mL of IL-1b, the final concentration of 1mg/mL of the final concentration of 1000U/mL of the IL-6, the PGE2.
It is special in preparation induction that the present invention also provides the DC cells of the gene modification obtained using said gene method of modifying
Application in specific immunological response drug.
In application of the present invention, preferably the DC cell infusion of the obtained gene modification is entered rich in lymph node
Oxter, it is primary every inoculation in 1 week, it carries out 3 times altogether, it is anti-that MHC-I class and II class in human dendritic cell (DCs) can be significantly increased
The expression of former epitope carries out antigen submission by MHC-I classpath, generates CD4+And CD8+It is special to repair patient for killer T cell
Specific humoral immune and cellular immunity, and then HPV16 infected tissue cell is killed, Long-term Infection is removed comprehensively
HPV16 virus, does not stay future trouble, prevents the generation of cervical carcinoma.
Offer the gene modification side of efficiency to raising DC cell HPV viruse antigen provided by the invention below with reference to embodiment
Method and its application are described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Using the fusion gene sequence of artificial synthesized method synthesis SEQ ID NO.1, while by the fusion gene sequence
It constructs into pGEM-T carrier, obtains pGEM-T-rHPV16-E6E7 plasmid, fusion is made to realize prokaryotic expression in host strain.
The in-vitro transcription process for starting the driving of T7 promoter, obtains the HPV16 mRNA molecule of improvement and is concentrated.
Infection HPV16 hypotype and development are obtained as the peripheric venous blood 50mL of CIN2 and CIN3, separation of lymphocytes is added
Liquid centrifugation layering, takes the cell of second layer mononuclearcell layer, cell is diluted with serum-free X-VIVO-15 culture medium,
Cell in 37 DEG C of 5%CO2It is incubated for 2.5 hours in incubator, obtains adherent mononuclearcell layer；It is thin that suspension is washed away with PBS
Born of the same parents are added and contain 800IU/mL granulocyte/macrophage colony stimulating factor (GM-CSF) and 500IU/mL rIL-4 X-
VIVO-15 culture medium culture 24 hours obtains immature Dendritic Cells (immature DC, iDC)；Every 3 days half amounts change liquid
Once, and GM-CSF and IL-4 cell factor is supplied；At the 6th day of culture, recombined human TNF- is added in X-VIVO-15 culture medium
(10ng/mL), IL-1b (10ng/mL), IL-6 (1000U/ml) and PGE2 (1mg/mL) induce DC cell maturation.
According to 1g/106The dosage of cell carries out electrotransfection.MRNA is placed in the electric revolving cup of sterilizing, with Gene Pulser
Xcell electroporation complete system carries out electrotransfection with 300V/150F.
Embodiment 2
The DC cell infusion of gene modification is entered and is rich in the oxter of lymph node in CIN2 and CIN3 phase patient, was connect every 1 week
Kind is primary, carries out 3 times altogether；In 4th week, 2mL peripheric venous blood is extracted, with the expression of ELISA method detection cell factor,
Concrete outcome is as shown in Figure 2 and Table 1:
The expression of 1. cell factor of table
As it can be seen that the expression of Blood Cytokines IFN, TNF and IL2 dramatically increase after DC vaccine inoculation,
The horizontal increase of middle IL2 is the most obvious.
Pass through Flow cytometry CD4+And CD8+The quantity of T cell, as shown in figure 3, showing that vaccinee is intracorporal
Immune system is activated, while the CD4 of vaccinee+And CD8+The quantity of T cell also dramatically increases.
The present invention provides a kind of raising DC cell HPV viruse antigens to offer the gene modification method and its application of efficiency,
The DC cell through gene modification obtained, can significantly increase MHC-I class and II class antigen in human dendritic cell (DCs)
The expression of epitope carries out antigen submission by MHC-I classpath, generates CD4+And CD8+It is special to repair patient for killer T cell
Property humoral immunity and cellular immunity, and then HPV16 infected tissue cell is killed, removes the HPV16 of Long-term Infection comprehensively
Virus does not stay future trouble, prevents the generation of cervical carcinoma, can be used for preparing prevention and treatment uterine neck cancer drug.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Hebei Jianhai Bio-chip Technology Co., Ltd.
<120>a kind of DC cell HPV viruse antigen that improves offers DC cell after the gene modification method and gene modification of efficiency
Using
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1298
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gccaccatgc tggttatggc gccgcgtacc gttctgctgc tgctgtctgc ggcgctggcg 60
ctgaccgaaa cctgggcgca ccaaaagaga actgcaatgt ttcaggaccc acaggagcga 120
cccagaaagt taccacagtt atgcacagag ctgcaaacaa ctatacatga tataatatta 180
gaatgtgtgt actgcaagca acagttactg cgacgtgagg tatatgactt tgcttttcgg 240
gatttatgca tagtatatag agatgggaat ccatatgctg tatgtgataa atgtttaaag 300
ttttattcta aaattagtga gtatagacat tattgttata gtttgtatgg aacaacatta 360
gaacagcaat acaacaaacc gttgtgtgat ttgttaatta ggtgtattaa ctgtcaaaag 420
ccactgtgtc ctgaagaaaa gcaaagacat ctggacaaaa agcaaagatt ccataatata 480
aggggtcggt ggaccggtcg atgtatgtct tgttgcagat catcaagaac acgtagagaa 540
acccagctgc atggagatac acctacattg catgaatata tgttagattt gcaaccagag 600
acaactgatc tctactgtta tgagcaatta aatgacagct cagaggagga ggatgaaata 660
gatggtccag ctggacaagc agaaccggac agagcccatt acaatattgt aaccttttgt 720
tgcaagtgtg actctacgct tcggttgtgc gtacaaagca cacacgtaga cattcgtact 780
ttggaagacc tgttaatggg cacactagga attgtgtgcc ccatctgttc tcagaaacca 840
catggagata cacctacatt gcatgaatat atgttagatt tgcaaccaga gacaactgat 900
ctctactgtt atgagcaatt aaatgacagc tcagaggagg aggatgaaat agatggtcca 960
gctggacaag cagaaccgga cagagcccat tacaatattg taaccttttg ttgcaagtgt 1020
gactctacgc ttcggttgtg cgtacaaagc acacacgtag acattcgtac tttggaagac 1080
ctgttaatgg gcacactagg aattgtgtgc cccatctgtt ctcagaaacc atcgttggta 1140
tcgttgcggg tctggcggtt ctggcggttg ttgttatcgg tgcggttgtt gcggcggtta 1200
tgtgccgtcg taaatcttct ggtggtaaag gtggttctta ctctcaggcg gcgtgctctg 1260
actctgcgca gggttctgac gtttctctga ccgcgtga 1298
Claims (10)
1. a kind of gene modification method for improving DC cell HPV viruse antigen and offering efficiency, comprising the following steps:
1) Fusion gene construction is obtained into recombinant plasmid pGEM-T-rHPV16-E6E7 into pGEM-T vector plasmid；It is described to melt
The sequence of gene is closed as shown in SEQ ID NO.1；
2) the recombinant plasmid pGEM-T-rHPV16-E6E7 is linearized through restriction enzyme SpeI, with obtained line
Property plasmid as template, start the in-vitro transcription process of T7 promoter driving, obtain the HPV16mRNA molecule of improvement；
3) DC cell is transfected using the HPV16mRNA molecule of the improvement, obtains the DC cell of gene modification.
2. gene modification method according to claim 1, which is characterized in that successively include: in the step 1) fusion
Kozak sequence, signal peptide, E6, E7 sequence of HPV16 hypotype and molecular transport signal.
3. gene modification method according to claim 1, which is characterized in that the system of the step 2) in-vitro transcription is 20 μ L
System, comprising: 2 × NTP/CAP, 10 μ L；
4. gene modification method according to claim 1, which is characterized in that further include described in concentration before the step 3) transfection
The HPV16mRNA molecule of improvement, the method for the concentration include: that 30 μ L nuclease frees are added into the reaction system of in-vitro transcription
Distilled water and 30 μ L lithium chloride precipitated liquids, -20 DEG C of 30~60min of placement；Under the conditions of 4 DEG C, it is heavy to collect RNA for centrifugation removal supernatant
It forms sediment；Using RNA precipitate described in TE buffer solution, carries out quantitative to RNA solution with ultraviolet specrophotometer and protected in -70 DEG C
It deposits.
5. gene modification method according to claim 4, which is characterized in that the lithium chloride precipitated liquid includes following content
Component: the EDTA of the LiCl and 50mM of 7.5M, the pH value of the lithium chloride precipitated liquid are 8.0.
6. gene modification method according to claim 1, which is characterized in that the method for the step 3) transfection is electrotransfection.
7. gene modification method according to claim 1, which is characterized in that the cultural method of step 3) the DC cell, packet
It includes: using the peripheric venous blood 50mL after infection HPV, adding the centrifugation layering of 25mL lymphocyte separation medium, take the single core of the second layer
The cell of cellular layer is diluted the cell using serum-free X-VIVO-15 culture medium, in 37 DEG C of 5%CO2In incubator
It is incubated for 2.5 hours, obtains adherent mononuclearcell layer；Suspension cell is washed away with PBS, be added containing 800IU/mL granulocyte/
The culture of X-VIVO-15 culture medium 24 hours of macrophage colony stimulating factor and 500IU/mL interleukin-4 cell factor, obtain
To immature Dendritic Cells；It changes the liquid once in half within every 3 days, and supplies granulocyte/macrophage colony stimulating factor and white
- 4 cell factor of interleukin；X-VIVO-15 culture medium be added recombination humanTNF-α, IL-1b, IL-6 and PGE2, induction DC cell at
It is ripe.
8. gene modification method according to claim 7, which is characterized in that the infection HPV is infection HPV16 hypotype and hair
Exhibition is CIN2 and CIN3 period.
9. gene modification method according to claim 7, which is characterized in that described in the X-VIVO-15 culture medium
The final concentration of 1000U/mL of the final concentration of 10ng/mL of the final concentration of 10ng/mL of TNF, the IL-1b, the IL-6, institute
State the final concentration of 1mg/mL of PGE2.
10. the DC cell of the gene modification obtained using any one of the claim 1~9 gene modification method is induced in preparation
Application in specific immune response drug.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111150716A (en) * | 2020-01-21 | 2020-05-15 | 厦门大学 | Universal antigen self-presenting tumor vaccine and preparation method thereof |
| CN111450244A (en) * | 2020-04-30 | 2020-07-28 | 北京翊博普惠生物科技发展有限公司 | Cell composition for preventing and treating coronavirus infection and application thereof |
| CN111748581A (en) * | 2019-09-23 | 2020-10-09 | 陕西众惠生物科技有限公司 | Method for transfecting dendritic cells by using hypoxia inducible factor recombinant expression plasmid |
| CN112891526A (en) * | 2020-08-18 | 2021-06-04 | 深圳市俊元生物科技有限公司 | HPV (human papillomavirus) virus vaccine based on personalized modified dendritic cells |
| CN112941030A (en) * | 2021-01-25 | 2021-06-11 | 吉林大学 | Antigen-specific Treg and preparation method and application thereof |
| CN114099662A (en) * | 2021-10-27 | 2022-03-01 | 山西协策企业管理咨询有限公司 | Monocyte-loaded E6E7 fusion protein vaccine composition and preparation method and application thereof |
| CN115960828A (en) * | 2023-02-01 | 2023-04-14 | 青岛海尔生物科技有限公司 | Preparation method of HLA-A0201 restrictive CD8 toxic T cells targeting HPV16E7 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181406A (en) * | 2011-03-02 | 2011-09-14 | 北京工业大学 | Recombinant adenovirus, dendritic cell (DC) modified by recombinant adenovirus and application of recombinant adenovirus and DC |
| CN104830781A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
-
2018
- 2018-11-12 CN CN201811338184.7A patent/CN109456945A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181406A (en) * | 2011-03-02 | 2011-09-14 | 北京工业大学 | Recombinant adenovirus, dendritic cell (DC) modified by recombinant adenovirus and application of recombinant adenovirus and DC |
| CN104830781A (en) * | 2015-05-05 | 2015-08-12 | 杨光华 | HSV-2 antigen based DC cell and targeting immune cell population, and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| MOHAMMAD SADRAEIAN ET AL.: "Prevention and inhibition of TC-1 cell growth in tumor bearing mice by HPV16 E7 protein in fusion with Shiga toxin B-subunit from shigella dysenteriae", 《CELL J》 * |
| 任会均: "HPV16E6基因转染人树突状细胞抗宫颈癌实验研究", 《中国优秀博硕士学位论文全文数据库（硕士）医药卫生科技辑》 * |
| 李丹等: "含HPV16 E6E7突变融合基因DC疫苗的抗肿瘤效果研究", 《中华实验和临床病毒学杂志》 * |
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| CN111150716A (en) * | 2020-01-21 | 2020-05-15 | 厦门大学 | Universal antigen self-presenting tumor vaccine and preparation method thereof |
| CN111150716B (en) * | 2020-01-21 | 2021-05-07 | 厦门宏谱福生物科技有限公司 | Universal antigen self-presenting tumor vaccine and preparation method thereof |
| CN111450244A (en) * | 2020-04-30 | 2020-07-28 | 北京翊博普惠生物科技发展有限公司 | Cell composition for preventing and treating coronavirus infection and application thereof |
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| CN112891526A (en) * | 2020-08-18 | 2021-06-04 | 深圳市俊元生物科技有限公司 | HPV (human papillomavirus) virus vaccine based on personalized modified dendritic cells |
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