CN109081873A - A kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines - Google Patents
A kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines Download PDFInfo
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- CN109081873A CN109081873A CN201810908222.1A CN201810908222A CN109081873A CN 109081873 A CN109081873 A CN 109081873A CN 201810908222 A CN201810908222 A CN 201810908222A CN 109081873 A CN109081873 A CN 109081873A
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Abstract
The invention discloses a kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines.The design scheme of the antitumor recombination NMM fused antigen are as follows: NY-ESO-1 is connected with 5 ' ends of MUCI gene by linking arm GCAGCATAT, 3 ' ends of MUCI gene are connected to 5 ' ends of MAGE-A3 encoding gene by linking arm AAGAAG.NMM fused antigen target spot of the present invention can cover kinds of tumors, especially some superficial tumors, such as breast cancer, lymthoma, cutaneum carcinoma, thyroid cancer and melanoma.Tumour Plasmid DNA vaccines pNMM of the invention is a kind of novel immune therapeutic agent that efficient system anti tumor immune response can be generated by locally injecting, and safety and tolerance are good, and having development is the potentiality that effective antitumour recurs drug.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to a kind of antitumor recombination NMM fused antigen Plasmid DNA epidemic disease
Seedling.
Background technique
Currently, tumour is one of an important public health problem and major causes of death of countries in the world.According to complete
It was predicted that by 2025, the whole world is annual newly-increased to suffer from carninomatosis example and will increase to 20,000,000 ball cancer epidemiology statistical number.It is reported that more
The cancer morbidity and the death rate in the U.S. Nian Lai are continuously declining.This shows to can achieve by modes such as medical procedures pair
The prevention and control of cancer.However in recent years, as the growth of population and aging degree aggravate, China's tumor patient disease incidence
With death rate Continued.Cancer is still the serious Disease Spectrum in China.Therefore, to tumour prevent, treat and prognosis
Research be still the most important thing.
The treatment of tumour occurs the 4th after simple operation excision, " fight the enemy 1,000 from damage 800 " radiotherapy/chemotherapy
Big oncotherapy technology-immunization therapy, i.e., by the method for targets neoplastic cells or activating immune cell, restarting is maintained
Immune system kills the identification of tumour cell, restores the normal anti tumor immune response of body.Si Long Caitlin Cancer center
Dispatch points out that following oncotherapy should be combineding with each other for immunization therapy, rather than single therapeutic modality.Checkpoint class
Monoclonal antibody, CAR-T are the representative means in current cancer immunization therapy, and there are many research institution and researcher's quantity.Although they
There are some therapeutic effects to tumour to a certain extent, but do not reach expected, and systemic administration can induce it is certain
Such as the adverse reaction of cytokine storm class, the application of its partial clinical and development are limited.
One of the hot spot of therapeutic tumor vaccine research in recent years, principle are that tumor target antigen delivery is entered human body,
Induction body actively generates specific immune response, prevents the growth, diffusion and recurrence of tumour, to reach removing or control tumour
Purpose, be a kind of new bio immunological technique drug.Immunization therapy with tumour specific antigen (TSA) can star with
Anti-tumor effect based on tumor-specific cytotoxicity T lymphocyte (cytotoxic Tlymphocyte, CTL) reaction, has
Effect strike tumour prevents transfer, recurrence and does not injure extraneous tissue, and antineoplastic specificity and immunological memory are other methods
It cannot compare.
Such drug can be by locally injecting, and safety and tolerance are fine, and can inducible system it is immune, be a kind of
Mild and effective tumor therapeutic agent.In the last decade time, the development speed of tumor vaccine is not quickly, with technology
Development, the especially development of high-flux sequence, individualized treatment and intratumor injection technology brings to the development of tumor vaccine
New power.It is 2 years especially nearly, the U.S., Liang Ge research group of Germany personalized tumor vaccine efficient clinical test results
Disclosure and Stanford University scholar shocking zoopery knot acquired by tumor vaccine delivered by intratumor injection mode
Fruit, and pushed the research of tumor vaccine to the teeth of the storm, win the concern of numerous research institutions and investment institution.It is therapeutic
Tumor vaccine is the important technical for preventing and treating the transfer of malignant tumour postoperative recurrence, is that the important industry of biomedicine field increases
Point.Utilize therapeutic vaccine carry out tumour extreme early intervention and prevention of postoperative recurrence, it is possible to for tumour prevention and control
Treatment brings new hope.
Summary of the invention
The purpose of the present invention is to provide a kind of antitumor recombination NMM fused antigen Plasmid DNA vaccines.
A kind of antitumor recombination NMM fused antigen, the antitumor recombination NMM fused antigen is with MAGE-A3 for main antigen
NY-ESO-1 antigens c TL epitope is merged in frame, fused upstream MUC1 antigens c TL epitope, downstream.
The design integration program of the antitumor recombination NMM fused antigen are as follows: by linking arm GCAGCATAT by NY-
ESO-1 is connected with 5 ' ends of MUCI gene, and 3 ' ends of MUCI gene are connected to MAGE-A3 encoding gene by linking arm AAGAAG
5 ' end.
The amino acid sequence of the antitumor recombination NMM fused antigen is as shown in sequence table SEQ ID NO:1.
The gene order of the antitumor recombination NMM fused antigen is as shown in sequence table SEQ ID NO:2.
Plasmid vector containing above-mentioned antitumor recombination NMM fusion antigen gene.
The host strain of plasmid vector containing above-mentioned antitumor recombination NMM fusion antigen gene.
Expand the primer of any segment in above-mentioned antitumor recombination NMM fusion antigen gene.
Above-mentioned antitumor recombination NMM fusion antigen gene is preparing the application in antitumor Plasmid DNA vaccines.
The tumour is breast cancer, lymthoma, cutaneum carcinoma, thyroid cancer and melanoma.
Beneficial effects of the present invention: the present invention, which designs and construct a kind of merge, 3 tumour antigen target spot (NY-ESO-
1, MUC1, MAGE-A3) recombinant plasmid dna (pNMM) vaccine, this 3 tumour antigen target spots can cover kinds of tumors, especially
It is some superficial tumors, such as breast cancer, lymthoma, cutaneum carcinoma, thyroid cancer and melanoma, above 5 kinds of tumours are annual
About 500,000 people of new cases, recombinant plasmid dna feature of the invention are tumour multiple target point, the covering of tumour polymorphic type.The recombination
Amalgamation and expression is gone back in Plasmid DNA can stimulate DC proliferation and mature FLT3L, CD40L, and panimmunity can be stimulated thin
The GM-CSF and costimulatory molecules CD80 that born of the same parents are proliferated, break up, theoretically can further improve the immunogene of the Plasmid DNA
Property, and then enhance its antineoplastic immune effect.PNMM can be immunized by way of intramuscular injection, can also be by tumor
Injection is immunized, and is speculated according to previous research, intratumor injection mode should can achieve more preferably effect.Meanwhile the vaccine
Both it can be used alone, the application of combination therapy can also be carried out with other immune formulations.Exclusive use can be played antitumor
The effect of recurrence and transfer, when being used in combination, other than it can enhance antitumous effect, can also reduce monoclonal antibody class or CAR-T
The dosage of para-immunity preparation, to greatly reduce the probability of systemic adverse reactions.Tumour Plasmid DNA vaccines pNMM of the invention is
A kind of novel immune therapeutic agent that efficient system anti tumor immune response can be generated by locally injecting, safety and
Tolerance is good, and having development is the potentiality that effective antitumour recurs drug.
Detailed description of the invention
Fig. 1 pUC57-NMM plasmid double digestion；In figure, before 1:pUC57-NMM plasmid enzyme restriction；2:pUC57-NMM plasmid Bgl
II and NotI double digestion；3:DNA marker.
Fig. 2 pVAX1-FLT3L-4P-CD40L-IRES-GM+B7 plasmid double digestion；In figure, 1:pVAX1-FLT3L-4P-
Before CD40L-IRES-GM+B7 plasmid enzyme restriction；2:pVAX1-FLT3L-4P-CD40L-IRES-GM+B7 plasmid Bgl II and NotI are bis-
Digestion；3:DNA marker.
The identification of Fig. 3 pVAX1-FLT3L-NMM-CD40L-IRES-GM+B7 plasmid double digestion；In figure, 1:pVAX1-
FLT3L-NMM-CD40L-IRES-GM+B7 plasmid Bgl II and NotI double digestion；2:DNA marker.
The identification of Fig. 4 pSFVK1-FLT3L-NMM-CD40L-IRES-GM+B7 (pSFVK1-NMM) plasmid double digestion；In figure,
Before 1:pSFVK1-NMM plasmid enzyme restriction；2:pSFVK1-NMM plasmid BamHI digestion；3:DNA marker.
The Western blot testing result of Fig. 5 pSFVK1-NMM plasmid expression antigen.
Fig. 6 ELISPOT statistic mixed-state result.
The tumor formation time of each experimental mice of Fig. 7 counts.
The tumor growth curve of each experimental group tumor-bearing mice of Fig. 8.
The average knurl weight of each experimental group tumor-bearing mice of Fig. 9.
Each experimental group tumor control rate of Figure 10.
Specific embodiment
The present invention will be further described in the following with reference to the drawings and specific embodiments.
Experiment is synthesized with primer segments by Sangon Biotech (Shanghai) Co., Ltd.；Carrier pSFVK1 and transition matter
By our company's building, (construction method refers to following patent: application number to grain pVAX1-FLT3L-4P-CD40-IRES-GM/B7
2015107650912)；The plasmid pUC57-NY-ESO-1/MUC1/ of fusion containing source of people NY-ESO-1, MUC1 and MAGE-A3
MAGE-A3 is synthesized and is constructed by Jin Weizhi company (Genewiz).50X TAE (Tris acetic acid running buffer) is rich from Beijing
Create Science and Technology Ltd.；Regular Agarose G-10 comes from Biowest company；Ttyptone and Yeast extract comes
From Oxoid company；Agar powder, purifying come from Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride and ethyl alcohol come from Beijing chemical industry
Factory；2x Taq PCR Mix wins Creative Technology Ltd. purchased from Beijing；Seamless Assembly Cloning Kit comes from
Sino-U.S. calm and peaceful biotechnology (Beijing) Co., Ltd；It is limited that competence reagent preparation box carrys out spontaneous work bioengineering (Shanghai) share
Company；A large amount of Ago-Gel DNA QIAquick Gel Extraction Kits (centrifugal column type) come from TIANGEN company；Small amount plasmid extraction kit
(centrifugal column type) and a large amount of extracts kits of plasmid (centrifugal column type) come from the east Beijing Hui Tian Science and Technology Ltd.；T4DNA connects
Enzyme is connect, DNA restriction enzyme is purchased from New England Biolabs (NEB) company；Gel reclaims kit, plasmid recycling
It is century Biotechnology Co., Ltd that kit, which is purchased from health,.
The design and expression plasmid of 1 fused antigen of embodiment construct
1. the analysis of antigen, selection and design
With MAGE-A3 for main antigen frame, NY-ESO-1 antigens c TL is merged in fused upstream MUC1 antigens c TL epitope, downstream
Epitope.NY-ESO-1 is connected with 5 ' ends of MUCI gene by linking arm GCAGCATAT first, 3 ' ends of MUCI gene pass through
Linking arm AAGAAG is connected to 5 ' ends of MAGE-A3 encoding gene, and final fusion overall length is 1073bp (such as sequence table SEQ
Shown in ID NO:2), encode 353 amino acid (as shown in sequence table SEQ ID NO:1).Its amino acid sequence such as the following table 1 (-
The linking arm that AAY- and-KK- is added):
The design of 1 fused antigen of table
Finally, the complete sequence of fusion antigen gene is synthesized that (sequence includes that upstream introduces a digestion position by Jin Weizhi company
Point BglII, downstream introduce restriction enzyme site NotI, thickened portion), complete sequence is as shown in sequence table SEQ ID NO:2.
2. obtaining NMM fusion antigen gene segment
After above-mentioned fusion antigen gene sequent synthesis, by digestion, purifying in the way of from plasmid pUC57-NY-ESO-1/
NMM fusion antigen gene segment NY-ESO-1/MUC1/MAGE-A3 is obtained in MUC1/MAGE-A3, digestion products carry out 1% fine jade
Lipolysaccharide electroresis appraisal.It is illustrated in fig. 1 shown below, NMM antigen gene fragments molecules amount size and expected consistent, about 1065bp.
The preparation and digestion of 2.1 transition plasmid pVAX1-FLT3L-4P-CD40L-IRES-GM+B7 is recycled
Plasmid, transition plasmid pVAX1- are extracted according to Beijing east Hui Tian Science and Technology Ltd.'s small amount plasmid extraction kit
FLT3L-4P-CD40L-IRES-GM/B7 generates two genetic fragments of different sizes, such as after II/Not of Bgl, I double digestion
Shown in Fig. 2.Large fragment is recycled, as the skeleton of building plasmid, referred to as transition vector pVAX1-FLT3L-CD40L-IRES-GM+
B7。
2.2 transition vector pVAX1-FLT3L-CD40L-IRES-GM/B7 and NMM fusion antigen gene NY-ESO-1/
MUC1/MAGE-A3T4 connection and conversion
The transition vector of above-mentioned gel extraction is connect with NMM fusion antigen gene, system is as follows:
Reaction condition 25 DEG C of connections 15min, 65 DEG C of 15min go connection enzymatic activity.
Connection product is transformed into E.coli.DH5 α competent E.coli, bacterium colony PCR identification clone:
Since DNA vector with fused antigen segment is connected by II/Not of Bgl I, design upstream F and downstream R's draws
The upstream and downstream of object fused antigen containing lid are as amplification object:
Upstream F:5 '-GGTACCGCCACCATGTCCCTGTTGATGTGGATC-3 '
Downstream R:5 '-CTCGAGTCATTACTCTTCCCCCTCTCTC-3 '
PCR system (12 μ L): 2 × PCR mix of 5ul；The ddH of 5ul2O；The R (10uM) of F (10uM) 1ul of 1ul is right
Connection product transformed bacteria drops into row PCR amplification.PCR is carried out with PCR instrument, reaction condition is 95 DEG C of 5min 1 circulations；94℃
30s, 55 DEG C of 30s, 72 DEG C of 30s expand 30 circulations；72℃7min.PCR product 0.8% agarose DNA gel electrophoresis point
Analysis.Qualification result is as shown in Figure 3.
The recombination of 2.3 carrier pSFVK1 and FLT3L-NY-ESO-1/MUC1/MAGE-A3-CD40-IRES-GM/B7 segment
Connection
Using pVAX1-FLT3L-NY-ESO-1/MUC1/MAGE-A3-CD40-IRES-GM/B7 plasmid as template, upstream is used
Primer 4pGMb7-r and downstream primer 4pGMb8-f are as follows:
4pGMb7-r:5 ' CGTTAATACACAGAATTCTGATTGGATCCCGGATCCGCCACCATGACAGTG3 '
4pGMb8-f:5 ' GCGTAGGGATGTAATTCAATTAATTACCCCTCGAGCGCGCTTATACAGGGCGTAC 3 '
Amplifying target genes DNA fragmentation carries out PCR amplification with PCR instrument, reaction condition, and amplification system is as follows:
Reaction condition is 95 DEG C of 5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min, 35 circulations；72℃7min.PCR product
With 0.8% agarose DNA gel electrophoretic analysis, gel extraction 4.3kb target gene fragment, recycling step is same as above.
Linearization process is carried out using Sma I single endonuclease digestion carrier, single endonuclease digestion system is as follows:
Through 25 DEG C digestion 4 hours, 65 DEG C of 20min, through 0.7% electroresis appraisal and purification and recovery.
Linearized vector pSFVK1 and target DNA FLT3L-NY-ESO-1/MUC1/MAGE-A3-CD40-IRES-GM/B7
Recombination connection to be gently mixed system as follows, be centrifuged the several seconds, reacted 15 minutes for 50 DEG C on heating instrument.
After reaction, centrifuge tube is placed on ice, progress bacterium conversion, the several monoclonal thallus of picking after conversion, into
Row PCR identification: what it is due to pSFVK1 carrier and target DNA is recombination connection, we are drawn with upstream primer 4pGMb7-r with downstream
The upstream and downstream of object 4pGMb8-f target DNA containing lid are as amplification object:
4pGMb7-r:5 ' CGTTAATACACAGAATTCTGATTGGATCCCGGATCCGCCACCATGACAGTG3 '
4pGMb8-f:5 ' GCGTAGGGATGTAATTCAATTAATTACCCCTCGAGCGCGCTTATACAGGGCGTAC 3 '
PCR system (12 μ L): 2 × PCR mix of 5ul；The ddH2O of 5ul；4pGMb7-r (10uM) 1ul's of 1ul
4pGMb8-f (10uM) drops into row PCR amplification to connection product transformed bacteria.PCR is carried out with PCR instrument, reaction condition is 95 DEG C
5min 1 circulation；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 2min expand 30 circulations；72℃7min.0.8% fine jade of PCR product
Lipolysaccharide DNA gel electrophoretic analysis.
Bacterium colony PCR is accredited as positive clone, extracts plasmid, carries out BamHI digestion identification (as shown in Figure 4).Final structure
It builds successful recombinant plasmid and is named as pSFVK1-NMM (pNMM).
The transfection of 2.4 recombinant plasmid dnas and expression identification
Transfection process
A. trypsin digestion collects 293T cell, is resuspended and is counted with fresh medium, every hole inoculation 20 in six orifice plates
Ten thousand cells are cultivated, and (the transfection same day) cell confluency degree reaches 50%-80% after 24h；
B. on the day of transfection, prepare transfection composite first: taking sterilizing EP pipe (1.5ml), the DMEM culture of 200 μ l is added
The Chemifect of base, the pSFVK1-NMM plasmid of 2 μ g and 6 μ l is mixed, and is stored at room temperature 30min, forms transfecting complexes；
C. the transfection composite after incubation is softly added in cell, is all around gently shaken, make it sufficiently
It mixes, marks, be put into 5%CO2, cultivate in 37 DEG C of cell incubator；
D.6h culture solution, one group while 5 μ l of addition of Z-VAD are replaced afterwards；
E. 48h after cultivating collects transfection cell, carries out Western Blot detection of expression.
Expression identification
(1) prepared by protein sample
A. the 293T cell after transiently transfecting 48h is washed twice with PBS；
B. the Laemmli Sample Buffer of 150 μ l is added, under cell scraper and will be transferred to cell scraper
In 1.5mlEP pipe；
C. it is put into dry-type thermostat, 100 DEG C of heating 10min lytic cells；
E. it is spare the sample after cracking to be placed in 4 DEG C of refrigerators.
(2) .SDS-PAGE electrophoresis
A. 10% separation gel (PH 8.8) is prepared:
B. it is 5.5cm that the glue prepared, which is slowly added into the plate assembled to Gel Height, and reserved 2cm height is matched
Set concentration glue；
C. prepare 5% concentration glue (PH 6.8): specific preparation method is shown in Table 5；
D., the concentration glue prepared is added to the upper layer of separation gel, insertion comb prepares loading hole；
E. after gelling to be concentrated is solid, the both sides for pinching comb straight up gently extract comb；
F. 5% mercaptoethanol and mixing is added before loading in sample is detectd, the protein sample of mixing is added to upper layer
It is concentrated in glue, 15 holes μ l/, while pre-dyed marker is added；
G. electrophoresis is carried out under constant pressure 100V, when bromophenol blue is in a line, adjusts voltage to 200V；
H. when bromophenol blue reaches the bottom of glue, electrophoresis can be stopped, carrying out transferring film.
(3) shifts trace
A. enough transfering buffering liquids are prepared full of transfer groove；
B. gel is removed from glass plate, glue is concentrated in removal upper layer；
C. gel is placed in transfering buffering liquid and impregnates 1min；
D. pvdf membrane is impregnated into 30s in methyl alcohol, guarantees that film becomes translucent；
E. pvdf membrane is put into equilibration buffer and impregnates 3-5min；
F. filter paper, gel, pvdf membrane are successively paved, as shown in Figure 1, avoiding the occurrence of bubble and fold；
G. constant current 200mA transfers 2h under ice-water bath.
(4) is immunoreacted
A. the pvdf membrane after transfer shaken at room temperature 1h in 5% skimmed milk power (TBST preparation) is placed in close；
B. after closing, film 3 times are washed with TBST, each 5min；
C.1:2000BSA diluted 4 DEG C of Ms mab MAGE-A3 antibody overnight incubations；
D. after primary antibody is incubated for, TBST washes film 3 times, each 5min；
E.1:5000 the goat anti-mouse IgG of the diluted horseradish enzyme label of 5% skimmed milk power is incubated at room temperature 2h；
F. after secondary antibody is incubated for, TBST washes film 3 times, each 5min.
(6) .ECL color developing detection destination protein
A. Tanon-5200 chemiluminescence imaging system is opened, is begun to use after temperature drops to 25 DEG C；
B. ECL colour developing A liquid and B liquid are mixed to get appropriate mixed liquor according to the ratio of A:B=1:1；
C. pvdf membrane taking-up is slightly dried, is placed on clean glass plate, mixed liquor is added dropwise in pillar location；
D. shooting and picture superposition process are carried out according to specification.
Western blot testing result such as Fig. 5 shows, fused antigen theoretical molecular weight is 92kDa, detected representation size with
It is expected that consistent (Z-VAD is inhibitors of apoptosis, and effect is to inhibit Apoptosis).
2 immunogenicity research of embodiment and antitumous effect analysis
A large amount of extraction and purifications are immune to use pSFVK1-NMM Plasmid DNA
Endotoxin-free plasmid needed for zoopery is referring to TIANGEN Biotech's " endotoxin-free plasmid
The operation of big extraction reagent kit " specification.
Experimental animal grouping and injected material explanation
The female C57BL/6 mouse of 6-8 week old is randomly divided into 7 groups, every group 10.Group and injected material are as follows:
Immunization strategy
Experiment mice carries out 3 immunity inoculations, and time interval is 14 days.It is immune to be injected using quadriceps muscle of thigh muscle and simultaneously
The mode for carrying out electric pulse stimulation carries out, and carries out multiple spot electric pulse in injection site with living gene electroporation immediately after injection
Stimulation, the condition of electric pulse stimulation are as follows: voltage 60V, burst length 50ms, pulse number are 1Hz × 6 time.Exempt from for experiment mice 3 times
After epidemic disease inoculation, 5 in every group of mouse are served only for carrying out immunogenicity detection, and other the 5 of every group are served only for tumor challenge experiment.
The secretion of ELISPOT method detection immune mouse spleen cell antigentic specificity IFN-γ
Separating immune mouse spleen lymphocyte suspension:
A. the 2nd week after final immunization, the neck that breaks puts to death each group immunized mice, is soaked in 75% ethyl alcohol and is put into after 5min
In the super-clean bench that ultraviolet radiation disinfection is crossed；
B. aseptic condition menisectomy takes out mouse spleen, is placed in 200 mesh cell sieves after spleen is shredded, then by cell
Sieve is put into the plate of 1640 culture mediums of serum-free, is as far as possible ground spleen with grinding rod uniform；
C. gently the lymphocyte in cell sieve is gone out with 1640 culture mediums of serum-free；
D. 5mL mouse lymphocyte separating liquid is added in centrifuge tube, by the obtained splenocyte suspension of step c along pipe
Wall is slowly added dropwise above lymphocyte separation medium and (is careful not to mix the two), and 2500rpm is centrifuged 30min at room temperature；
E. it takes liquid to be divided into three layers after being centrifuged, takes middle layer tunica albuginea, 1640 culture mediums that 10mL serum-free is added clean lymph
Cell 2 times (1500rpm/min, 10min)；It is resuspended in 10mL serum free medium and counts.
It is grasped according to up to the Mouse IFN-γ precoated ELISPOT kit that section is Bioisystech Co., Ltd
Make, specific as follows:
First day: stimulant was added in inoculating cell, cultivates (rigorous concentration sterile working)
1. the activation of pre-coated plate: 200 μ L EZ-CultureTM serum free mediums or RPMI-1640 is added in every hole
Culture medium is deducted after being stored at room temperature 5-10min.
2. cell suspension is added: each experimental port, 100 μ L/well are added in the cell suspension for adjusting concentration.
Positive control hole: 1 × 105cells/well can be used in cell concentration；Negative control hole: cell concentration can be used 1 ×
105cells/well；Background negative control: be added be resuspended cell with culture medium (EZ-CultureTM without
Blood serum medium or RPMI-1640 culture medium containing fetal calf serum)；Experimental port: sample cell concentration is by experimenter
It is voluntarily adjusted according to experiment.
3. stimulant is added: 10 μ L/well, it is specific as follows: positive stimulus agent working solution positive control hole: is added.
Negative control hole: EZ-CultureTM serum free medium (or cell culture medium is resuspended) is added；Experimental port: it is added
The stimulant (being configured to 10 × final concentration with EZ-CultureTM serum free medium or RPMI 1640) of experimenter oneself.
4. being incubated for: after all samples and stimulant add, covering plate lid.37 DEG C are put into, 5%CO2 incubator culture 16-
20hr.Second day: operating (no longer needing sterile working) after culture
1. lytic cell: pouring aperture inner cell and culture medium.Cold deionized water on the rocks, 200 μ L/well, 4 DEG C of refrigerators are put
Set 10min hypotonic lysis cell.
2. board-washing: liquid in pouring aperture, 1 × Washing buffer, 200 μ L/well are washed 5-7 times.It stops every time
30-60s.For the last time, it is buckled on blotting paper dry.
3. detecting antibody incubation: each experimental port, 100 μ L/ are added in the antibody working solution of the biotin labeling diluted
well.37 DEG C of incubation 1hr.
4. board-washing: liquid in pouring aperture, 1 × Washing buffer, 200 μ L/well are washed 5 times.30- is stopped every time
60s.For the last time, it is buckled on blotting paper dry.
5. enzyme-linked Avidin is incubated for: each experimental port, 100 μ L/well are added in the enzyme mark Avidin working solution diluted.37
DEG C be incubated for 1hr.
6. board-washing: liquid in pouring aperture, 1 × Washing buffer, 200 μ L/well are washed 5 times.30- is stopped every time
60s.For the last time, it is buckled on blotting paper dry.
7. colour developing: each experimental port, 100 μ L/well are added in the AEC developing solution working solution now matched.Room temperature, which is protected from light, stands 15-
45min (at 20-25 DEG C, colour developing 25min is appropriate).If room temperature is lower than 20 DEG C, it is proposed that do and develop the color in 37 DEG C of incubators, every 5-
10min checks primary.
8. color development stopping: liquid in pouring aperture opens board bottom seat, with deionized water/originally water washing front and back sides and pedestal
3-5 times, color development stopping.Plate is placed on room temperature shady place, closes pedestal after natural drying to it.
9.ELISPOT plate spot count, and the various parameters of spot are recorded, it statisticallys analyze.
The proteantigen purified using early period as differential stimulus object, sterile isolated each group is immunized the leaching of mouse simultaneously
Bar cell is stimulated and is activated again, has the T lymphocyte of reaction to be activated specific antigen, starts secretion of gamma-IFN, this
A little cell factors are captured by the pre-coated IFN-γ monoclonal antibody on elisa plate, become spot by way of enzyme-linked colour developing
Point, and will not then be stimulated to the responseless lymphocyte of specific antigen and discharge cell factor, as a result as shown in Figure 6.
It can be seen that E group pSFVK1-NY-ESO-1/MUC1/MAGE-A3-IRES-GM/B7 plasmid, F group pSFVK1- from Fig. 6 result
FLT3L-NY-ESO-1/MUC1/MAGE-A3-IRES-GM/B7 plasmid and G group pSFVK1-FLT3L-NY-ESO-1/MUC1/
MAGE-A3-CD40L-IRES-GM/B7 plasmid, the i.e. experiment mice of pSFVK1-NMM can detect t cell immune response.Its
The spot number of middle G group is 1.8 times of the spot number that E group and F group induce generation with other groups compared to having significant difference
With 1.3 times.Should the experimental results showed that, fusion have the overall length plasmid pSFVK1-NMM of immunologic adjuvant molecule FLT3L and CD40L can
It induces immunized mice to generate stronger cell immune response, Plasmid DNA is exempted to demonstrate immunologic adjuvant molecule
The facilitation of epidemic focus.
Stablize the foundation of the tumor models of expression NMM antigen
In order to complete the drug efficacy study of recombinant plasmid, the tumor models of high expression NMM target antigen are established.Using base
Because synthetic method synthesizes MUC1, MAGE-A3, NY-ESO-1 fusion, is directed it and be inserted into very using DNA recombinant technique
In nuclear expression carrier pIRES-neo, using cationic polymer transfection B16 cell, is pressurizeed and screened by G418 drug, obtain energy
It is enough to stablize murine melanoma (B16) cell strain for expressing these three antigens, for the tumor-inhibiting action mechanism for evaluating recombinant plasmid dna
Cell model is provided, B16-NMM+ is named as.Meanwhile it being inoculated with C57BL/6 mouse using above-mentioned cell strain B16-NMM+, observation is steady
Surely the tumorigenesis ability of the B16 cell after foreign gene is transfected.
By measuring the tumor size of tumor-bearing mice, and depict each experimental mice tumor growth curve.As a result such as
Shown in Fig. 7 (tumor formation time) and Fig. 8 (tumor growth curve).A, B, C group mice-transplanted tumor tumor formation time is without marked difference, D, E,
F, the mouse of G group is extended (Fig. 7) than control group without tumor incubation period；D, remaining each control of the growth fraction of E, F, G group tumour
Group delays, and G group, the i.e. mouse of pNMM Plasmid DNA group are without tumor prolongation of latency time longest, growth fraction D, E, F group of tumour
Obviously delay (Fig. 8).
Tumor challenge experiment
The 7th day after each group mouse final immunization, with the tumor cell line for stablizing expression NMM antigen obtained in 2.7.6
Mouse is attacked.
Operating procedure are as follows: C57BL/6 mouse carries out continuous 3 vaccine inoculation before carrying out transplantable tumor attack, at interval of 14
It is 1 time immune, immune to be injected using quadriceps muscle of thigh muscle and simultaneously carry out by the way of electric pulse stimulation, with ECM830 electricity arteries and veins
It rushes introducing apparatus and carries out multiple spot electro photoluminescence, impulsive condition are as follows: voltage 60V, burst length 50ms, pulse number are in injection site
1Hz × 6 time.The progress transplantable tumor attack in 7 days after final immunization, dorsal sc are inoculated with 1*105A B16-NMM+ cell.It is transplanting
30-35 days disconnected necks put to death every group of 5 mouse after tumor attack, and operation strips tumor tissues and records various data.
The observation of DNA vaccination antitumor activity
After the attack of subcutaneous transplantation tumor, the growing state of mouse interior tumor is observed.When accessible tumor nodule, record is each
The tumor formation time of group mouse；Then, with vernier caliper measurement and the vertical major diameter of transplantable tumor and vertical short was recorded at interval of two days
Diameter calculates transplantable tumor volume according to formula, draws the growth curve of transplantable tumor in Mice Body；To growth of transplanted human to certain number of days
When, the neck that breaks puts to death every group of each 5 mouse, and operation strips tumor tissues and weighs knurl weight, according to the inhibiting rate of formula meter tumour.It is public
Formula is as follows:
Gross tumor volume (mm3)=0.5 × vertical major diameter × (vertical minor axis) 2；
Inhibition rate of tumor growth (%)=(blank group average knurl weight-experimental group average knurl weight)/blank group average knurl weight ×
100%.
As a result see Fig. 9 and Figure 10: wherein there was no significant difference (p > 0.05) for the average knurl weight of A, B, C group；With control group phase
Than D, E, F, G group average knurl weight are substantially reduced；The average knurl weight of G group mouse is minimum, and inhibition rate of tumor growth reaches
85.0%, it can be seen that the ability with strong antitumor attack after mouse is immunized in this group of plasmid, i.e. pNMM, this is for anti-swollen
Tumor recurrence is of great significance.
In order to verify the pNMM with strongest immunogenicity, the Plasmid DNA of several intermediate releases is also constructed, that is, is contained
The truncated-type control plasmid of part or all of stimulation molecule.After different plasmid DNA injection experimental animals, it can be seen that pNMM group
Strongest cell immune response can be induced, compared with other primary comparison groups, there is significant difference, this, which is able to demonstrate that, exempts from
Epidemic disease activation and stimulation molecule are for the importance of enhancing cell immune response.It is consistent with this, it can be seen that by attacking tumor experiment
PNMM Plasmid DNA group has strongest inhibition tumor growth effect, and the inhibition rate of tumor growth of the group can reach 85%, effect
Significantly.
Result above, the recombinant plasmid pNMM that can confirm building have the ability of very strong inhibition tumour growth, should
Kind is currently ongoing examination and preclinical study, and having development is the immunotherapy medicaments new product of effective antitumour recurrence
The prospect of kind.Meanwhile in further research, recombinant plasmid pNMM can be administered by we in a manner of intratumor injection,
Mainly for superficial tumor, tumor killing effect can be theoretically greatly enhanced.
Claims (9)
1. a kind of antitumor recombination NMM fused antigen, which is characterized in that the antitumor recombination NMM fused antigen is with MAGE-A3
For main antigen frame, fused upstream MUC1 antigens c TL epitope, NY-ESO-1 antigens c TL epitope is merged in downstream.
2. antitumor recombination NMM fused antigen according to claim 1, which is characterized in that the antitumor recombination NMM fusion
The design integration program of antigen are as follows: NY-ESO-1 is connected with 5 ' ends of MUCI gene by linking arm GCAGCATAT, MUCI base
3 ' ends of cause are connected to 5 ' ends of MAGE-A3 encoding gene by linking arm AAGAAG.
3. antitumor recombination NMM fused antigen according to claim 1, which is characterized in that the antitumor recombination NMM fusion
The amino acid sequence of antigen is as shown in sequence table SEQ ID NO:1.
4. antitumor recombination NMM fused antigen according to claim 1, which is characterized in that the antitumor recombination NMM fusion
The gene order of antigen is as shown in sequence table SEQ ID NO:2.
5. the plasmid vector containing antitumor recombination NMM fusion antigen gene described in claim 1.
6. the host strain containing the plasmid vector of antitumor recombination NMM fusion antigen gene described in claim 1.
7. expanding the primer of any segment in antitumor recombination NMM fusion antigen gene described in claim 1.
8. antitumor recombination NMM fusion antigen gene described in claim 1 is preparing the application in antitumor Plasmid DNA vaccines.
9. tumour recombination NMM fusion antigen gene is preparing answering in antitumor Plasmid DNA vaccines according to claim 8
With, which is characterized in that the tumour is breast cancer, lymthoma, cutaneum carcinoma, thyroid cancer and melanoma.
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| US6291430B1 (en) * | 1997-09-12 | 2001-09-18 | Ludwig Institute For Cancer Research | Mage-3 peptides presented by HLA class II molecules |
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