CN103409451A - Method for loading tumor antigen peptide to dendritic cell (DC) in targeting manner - Google Patents
Method for loading tumor antigen peptide to dendritic cell (DC) in targeting manner Download PDFInfo
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Abstract
The invention provides a method for loading a tumor antigen peptide to a dendritic cell (DC) in a targeting manner, and belongs to the field of medicine technology. The method is characterized in that a fusion protein with antibody activity is tightly coupled with a DC surface receptor molecule DEC-205, and a tumor antigen peptide MUC1 is loaded to the DC in a targeting manner, so that the capacity of the DC in terms of the presentation of the tumor antigen peptide MUC1 can be greatly improved.
Description
Technical field
The invention belongs to field of medical technology, particularly in the knubble biological technology, be used for the treatment of the method that the tumour antigen target loads.
Background technology
Tumor biotherapy is the 4th class cancer treatment method developed after operation, radiation and chemotherapy, is to utilize the immune response that body excites to resist, suppress and kill cancer cells.Different from traditional methods for the treatment of, tumor biotherapy is to take the front line sciences such as modern molecular biology, cytobiology and molecular immunology to be basis, utilize the immunne response of body to tumour, by biological means, change the interaction between tumour and body, transfer the defense mechanism of body to tumour, reach the control tumor growth, extend the survival of patients phase, improve the purpose of patients ' life quality, it has become the critical treatment method in combined therapy of tumour.
Immunity system is the defense system of human body, removes the external foreign matters such as bacterium, virus on the one hand, removes on the other hand nonfunctional cell old and feeble in body and the cell of undergoing mutation.Mutant cell further becomes tumour cell, and the human immune system can identify these tumour cells in time, and is removed.Body immune system realizes that this removing function need have two important links, the one, and identification (tumour antigen identification), the 2nd, kill and wound (removing tumour cell).The identification link mainly depends on the cells such as dendritic cell (DC) with antigen presentation function, kills and wounds link and relies on the multiple immunological lymphocyte of removing function that has.The DC cell obtains the key that tumour antigen is the immune system specific tumor cell.
The research of process recent two decades and perfect, the vitro differentiation of DC cell and Activiation method have been tending towards ripe.But the tumour antigen loading technique of DC is also reached common understanding far away.Therefore, the difference key of different DC vaccine curative effects is selection and the antigen loading regime of tumour antigen.Domestic antigen loading method commonly used is that tumour homogenate is mixed and gets final product with DC.The hazardness of operation is like this, and tumour antigen belongs to self antigen, and the simple and inefficient self antigen loaded not only can not the induction of immunity reaction, can cause on the contrary immune stagflation, and the result is just the contrary.
Summary of the invention
The purpose of this invention is to provide a kind of method to dendritic cell target loading tumor antigen peptide that can overcome above defect.
The present invention utilizes gene vitro recombination and the expression of antibody gene and the codes for tumor antigen peptide MUC1 of anti-DEC-205 molecule, the fusion rotein V of acquisition and the efficient combination of dendritic cell DC surface DEC-205 acceptor molecule, obtain the tumor antigen peptide MUC1 that the dendritic cell target is loaded.
Concrete steps are:
1) will resist the antibody gene of DEC-205 and the gene of coding MUC1 synthesize and recombinate external, obtaining preserving number CGMCC is the expression plasmid pET41a-antiDEC-ScFV of NO:7686;
2) expression plasmid pET41a-antiDEC-ScFV is proceeded to intestinal bacteria, through inducing, obtain expressed fusion protein V precursor;
3) by fusion rotein V precursor after denature and renature, obtain the fusion rotein V with active function;
4) adopt biotin labeled fusion rotein V with active function to be combined with dendritic cell DC, with the fluorescently-labeled affine tumor antigen peptide MUC1 that the dendritic cell target is loaded that usually detects.
A kind of method that the present invention adopts target to load, load tumor antigen peptide MUC1 to ripe DC cell surface.Ripe DC cell surface with antigen presentation function has a kind of distinctive acceptor DEC-205, therefore design the single-chain antibody gene of a kind of anti-DEC-205, and synthesize and recombinate external with the gene of codes for tumor antigen peptide MUC1, then at expression in escherichia coli, obtain the fusion rotein precursor; The fusion rotein precursor, after renaturation, becomes the fusion rotein V with antibody activity.This protein molecular has the single-chain antibody of anti-DEC-205 at the N end, at the C end, contain tumor antigen peptide MUC1 simultaneously; After utilizing biotin labeling fusion rotein V, with ripe DC Cell binding and flow cytometer, detect again, through adopting flow cytometer to detect fusion rotein V antibody activity, confirm that fusion rotein V can be loaded into tumor antigen peptide MUC1 target ripe DC cell surface, can effectively improve the ability of DC cell submission tumour antigen.
Employing loads the method for tumor antigen peptide to the DC cell-targeting, submission tumour antigen especially efficiently, bring out body for tumour antigen immune response very efficiently, can transfer, activate and increase and have the killer cell (CD8+T cell) of cancer target, simultaneously can produce long-lasting memory cell, to reach the antitumor of long-term, sustainability, the effect of anti-recurrence.
The invention has the beneficial effects as follows: the inventive method adopts fusion rotein and DC cell surface receptor molecule DEC-205 with antibody activity to combine closely, to the DC cell-targeting, load tumor antigen peptide MUC1, can greatly improve the ability of DC cell submission tumor antigen peptide MUC1.
The purpose of above step 3) sex change or meaning, renaturation purpose or meaning: fusion rotein V of the present invention adopts prokaryotic organism e. coli protein expression system, the space structure that expressed hypoproteinosis is correct.Denaturation process is to affect the disulfide bond reduction of albumen space structure, then under given conditions again by the albumen slow oxidation, recovers disulfide linkage and correct albumen space structure, obtains the fusion rotein V with anti-DEC-205 activity.
In described step 1), the antibody molecule of anti-DEC-205 and tumor antigen peptide MUC1 are binned in same fusion rotein.The antibody activity that can keep anti-DEC-205.
In described step 2) in, the nucleotide sequence by the genosome of the antibody gene of anti-DEC-205 molecule and codes for tumor antigen peptide MUC1 reassembles into outward, be cloned on protein expressing plasmid, through inducing at expression in escherichia coli.
The accompanying drawing explanation
Fig. 1 is the design of graphics of expression plasmid pET41a-antiDEC-ScFV.
Fig. 2 is the SDS-PAGE electrophoresis evaluation figure of expression of recombinant proteins.
Fig. 3,4,5,6,7 is respectively the facs analysis figure that the fusion rotein V of blank group, negative control group 1, negative control group 2, test sample book and positive controls is combined with DC cell surface DEC-205 acceptor molecule.
Embodiment
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to corresponding raw material manufacturer.
One, the preparation of fusion rotein V and biotin labeling:
1, recombinant gene expression vector builds:
The antibody molecule of anti-DEC-205 and tumor antigen peptide MUC1 are binned in to the concrete measure in same fusion rotein:
According to function signal and corresponding nucleotide sequence, adopt the method for commercialization synthetic nucleic acid, 72 bases of synthetic 744 bases with antibody molecule (antiDEC-ScFV) of anti-DEC-205, MUC1 polypeptide (MUC1) and 69 bases of the needed functional zone of expression and purification (TEV, Myc, His6).Synthetic full gene fragment (855 bases) is cloned between upper two the restriction enzyme site Nde I of commercialization plasmid vector pET41a (+) and Xho I.And by the method for gene sequencing, checking recombination sequence and plasmid construction.
The design of graphics of expression plasmid pET41a-antiDEC-ScFV as shown in Figure 1, its function schematically as follows:
The nucleotide sequence of expression plasmid pET41a-antiDEC-ScFV:
atgcaggctg tggtgactca ggagagtgcc ctgaccacat cacctggcga gacagtgact 60
ctgacctgtc ggagctccac cggggcagtg acaatcagca actacgccaa ttgggtccag 120
gaaaaaccag accacctgtt cacagggctg attggcggga ctaacaatcg agcacccgga 180
gtgcctgcca ggtttagtgg gtcactgatc ggagacaagg ccgctctgac aattactggc 240
gcccagaccg aggatgaagc tatctacttc tgtgcactgt ggtataacaa tcagttcatt 300
tttgggagcg gaacaaaagt gactgtcctg ggcggcggag gaggaagcgg aggaggaggg 360
tccggaggcg ggggatctgg aggaggagga agtgaggtgc agctgcagca gtctggccct 420
gtgctggtca agccaggggc tagtgtgaaa atgtcatgca aggcaagcgg caacaccttc 480
acagacagct ttatgcactg gatgaaacag tcccatggaa agtctctgga gtggatcggc 540
atcattaacc cctacaatgg gggaacctcc tataatcaga agttcaaagg caaggccact 600
ctgaccgtgg ataaatctag ttcaaccgct tacatggagc tgaacagcct gacatccgaa 660
gactctgccg tgtactattg tgctcgcaat ggcgtccgat actattttga ttattggggc 720
caggggacta ccctgacagt gagcacagca cctccagctc atggtgtgac tagcgctcca 780
gatacccgcc cggcacccgg gtctacggcg cctccggaaa acctgtattt tcagggcgaa 840
cagaagctga ttagcgaaga ggatctgcac catcatcacc accat
The protein sequence of expression plasmid pET41a-antiDEC-ScFV:
MQAVVTQESALTTSPGETVTLTCRSSTGAVTISNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYNNQFIFGSGTKVTVLGGGGGSGGGGSGGGGSGGGGSEVQLQQSGPVLVKPGASVKMSCKASGNTFTDSFMHWMKQSHGKSLEWIGIINPYNGGTSYNQKFKGKATLTVDKSSSTAYMELNSLTSEDSAVYYCARNGVRYYFDYWGQGTTLTVSTAPPAHGVTSAPDTRPAPGSTAPPENLYFQGEQKLISEEDLHHHHHH
The expression plasmid pET41a-antiDEC-ScFV that the present invention builds, in on 06 08th, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preserving number: CGMCC NO:7686, Classification And Nomenclature: large intestine Erichsen bacterium
Escherichia coli, its length is: 5905 bases.
Separately, the single-chain antibody gene of anti-DEC-205 is published, and adopting the synthetic nucleic acid fragment is the commercial means commonly used in bioengineering field, as long as there is nucleotide sequence just can order nucleic acid fragment.
2, expression vector plasmid pET41a-antiDEC-ScFV conversion intestinal bacteria carry out protein expression:
The expression vector plasmid pET41a-antiDEC-ScFV that order-checking is correct imports Host Strains e. coli bl21 (DE3) with calcium chloride transformation, add 37 ℃ of concussions of SOC substratum to cultivate after 30 minutes, coat (final concentration is the 50ug/ml sulphuric acid kanamycin) in the LB flat board overnight incubation.Next day, bacterium colony in good condition on picking LB flat board, be inoculated in the LB substratum of sulfur acid kantlex.37 ℃ of shaking tables were cultivated after 7 hours, by 3ml bacterium liquid/L, were inoculated in 4L self-induction substratum TB 37 ℃ of shaking table overnight incubation.4000rpm, it is standby that 10 minutes centrifugal supernatants of abandoning, thalline are stored in-20 ℃ of refrigerators.
Use 12% polyacrylamide gel electrophoresis to identify protein expression, as shown in Figure 2, confirm that the fusion rotein precursor of expressing is expressed with the inclusion body form.Wherein, T: induce rear full bacterium lysate; S: induce rear full bacterium centrifugal supernatant after ultrasonication; P: the inclusion body after washing.
3, the sex change of fusion rotein precursor, renaturation and purifying:
Extract :-20 ℃ of 45 gram thalline that save backup are placed in to the broken bacterium damping fluid of 450ml CRB (by 5% glycerine, 50mM Tutofusin tris-hydrochloric acid, 50mM sodium-chlor, 0.5mM disodium ethylene diamine tetraacetate and 1mM tri-(2-propyloic) phosphine, the pH value is 8.0) in, magnetic agitation is even, the broken thalline of high pressure homogenate 5 times, more than operate in ice bath and carry out; Add NSDB-201 in broken bacterium liquid to final concentration 125mM, stirring at room 15 minutes, 9500 turn, 10 ℃ centrifugal 15 minutes, abandon supernatant; The centrifugation inclusion body is resuspended in 450ml IBWB damping fluid, stirring at room 15 minutes, 9500 turn, 10 ℃ centrifugal 15 minutes, abandon supernatant, repeated washing once, finally obtains the 2.3g inclusion body, frozen standby in-80 ℃.
Sex change: the 1g solubilization of inclusion bodies (is comprised of 7M Guanidinium hydrochloride, 50mM Tutofusin tris-hydrochloric acid, 0.2M sodium-chlor, 2.0mM disodium ethylene diamine tetraacetate and 10mM TCEP) in 20ml GDB damping fluid, 4 ℃ of soft stirrings are spent the night.Renaturation: above-mentioned 20ml is contained to dropwise joining in the 5L renaturation solution of fusion rotein and (formed by 0.5M arginine, 100mM Tutofusin tris-hydrochloric acid, 4mM disodium ethylene diamine tetraacetate, 240mM sodium-chlor, 1mM Repone K, 9mM reduced glutathion and 1mM Sleep-promoting factor B, the pH value is 9.0) to final concentration 200ug/ml, 4 ℃ of soft stirrings are spent the night.
The ultra-filtration membrane bag is used successively to the 0.1M sodium hydroxide of 2L, the apyrogenic ultrapure water of 2L, the 2Ltris/Urea damping fluid (is comprised of 50mM Tutofusin tris-hydrochloric acid and 150mM urea, the pH value is 8.0) clean, the 5L renaturation solution is concentrated into to 300ml, and continuing ultrafiltration and concentration is that the Tris/Urea damping fluid is to final volume 100ml, 20000g by buffer exchange, 4 ℃ of centrifugal 15min remove precipitation, and the 0.2um filter filters and is placed in 4 ℃ of preservations so that next step purifying.
Purifying: the fusion rotein solution example that the previous step renaturation is good is splined on the 10ml Q-FF anion-exchange column that balance is good, use buffer A (the 50 mM Tris of 2 column volumes, pH 8.0) and 5% buffer B (the 50 mM Tris of 5 column volumes, 1 M NaCl, pH 8.0) rinse near baseline, the linear gradient elution of 10 times of column volumes of 5%-100% B, finally use 5 times of column volumes, 100% buffer B wash-out.After collecting component 3-8, be 10kDa dialysis tubing dialysed overnight with molecular weight cut-off, sample is placed on-80 ℃ with the filtration of 0.2um filter and saves backup.Through determination of protein concentration, fusion rotein V ultimate capacity: 12.46 milligrams.
4, vitamin H (Biotin) mark of the fusion rotein V after purifying:
Get 2.8 milligrams of fusion rotein V by 0.1 mol/L sodium bicarbonate buffer liquid (pH 8.0) dialysed overnight; With 1ml DMSO, dissolving 1mg N-hydroxy-succinamide vitamin H (NHSB) concentration is 1mg/ml; To containing in fusion rotein V solution, add NHSB solution (ratio of fusion rotein V and NHSB is 8:1 (mol ratio), standing 2 hours of room temperature; Add again 24 microlitre 1 mol/L NH
4Cl (every 25 μ g NHSB add 1 μ l) reacts with unnecessary NHSB, at room temperature stirs 10 minutes; At 4 ℃, with PBS, fully dialyse, to remove free vitamin H, obtain biotin labeled fusion rotein V.
Two, the fusion rotein V of detection of biological element mark and ripe DC Cell binding efficiency:
1, peripheral blood mononuclear cell (PBMC) gathers preparation:
100 milliliters of the whole bloods of use Healthy People, with physiological saline, dilute by 1 ︰ 1 (volume ratio), proportion is that 1.077 lymphocyte separation medium Ficoll-Paque Plus and diluted blood add in centrifuge tube by 1:2, centrifugal 1500rpm/30min, get the interfacial layer cell, PBS washing 2 times, be resuspended in appropriate GT-T551 serum free medium.
2, monocyte separates, immature DC is cultivated and ripe DC induces:
A. get 1.5 * 10
8Cell, to the T150 Tissue Culture Flask, adds GT-T551 serum-free culture to 20 ml, puts 37 ℃, cultivates 2 hours in 5% CO2 incubator;
B. suck cell culture fluid, add 20 ml physiological saline washing 2 times;
C. in Tissue Culture Flask, add 25 ml to contain the serum-free DC substratum of final concentration 1000U/ml GM-CSF, 1000U/ml IL-4, put 37 ℃, cultivated 4 days in 5% CO2 incubator.
D. in Tissue Culture Flask, add the TNF-α of GM-CSF, the 50ng/ml of final concentration 1000U/ml, the PGE2 of 1nmol/ml, put 37 ℃, in 5% CO2 incubator, continue to cultivate 2 days.
3, ripe DC cell harvesting:
A. cell is laid from Tissue Culture Flask wall blowing up, go in 50 ml centrifuge tubes, centrifugal 10 minutes of 1500rpm, remove supernatant;
B. add the resuspended washing of 10 ml physiological saline, centrifugal 5 minutes of 1500rpm, abandon supernatant;
C. add 1 ml PBS resuspended, go in 1.5 ml centrifuge tubes, centrifugal 5 minutes of 1500rpm;
D. abandon supernatant, with 500 μ l, contain the PBS re-suspended cell of 1% BSA, the cell that takes a morsel adds trypan blue counting, cell density 6.45 * 10
6/ ml, Cell viability 93%.
4, with flow cytometer (FACS), analyze the joint efficiency of recombinant protein V and ripe DC cell surface receptor:
Need the molecule of identifying: recombinant protein V, first use vitamin H (Biotin) mark, concentration 0.403 μ g/ μ l; Then use the second antibody PE Streptavidin(avidin with fluorescence, concentration 0.2 μ g/ μ l) detect; Positive control adopts commercialization antibody (Peprotech), and band fluorescence PE mark (concentration 50ng/ μ l) is the same as with recombinant protein V the same acceptor on ripe DC cell.
By cell suspension 50 μ l/ pipes (3.22 * 10
5Cell) divide and install in 5 1.5 ml centrifuge tubes; One pipe is blank, residue 4 pipes add respectively the biotin labeled recombinant protein V(of 3 microlitre negative control 1), 6 microlitre PE-Streptavidin(negative controls 2), the biotin labeled recombinant protein V of 3 microlitre and 6 microlitre PE-Streptavidin(test sample books), the commercialization antibody (positive control) of 10 microlitre PE marks; The flow cytometer loading, carry out the FACS analysis.
As shown in Fig. 3,4,5,6,7, be respectively the facs analysis figure that the fusion rotein V of blank group, negative control group 1, negative control group 2, test sample book and positive controls is combined with DC cell surface DEC-205 acceptor molecule.
Conclusion: the FACS figure of test sample book and positive control shows the fluorescently-labeled albumen of PE and ripe DC Cell binding (R3 zone).In test sample book, there is 61.09% ripe DC cell to be combined with recombinant protein V, 75% ripe DC cell and commercialization antibodies arranged in positive control.Therefore, recombinant protein V and ripe DC cell surface receptor joint efficiency are 81.45% of commercialization antibody, and the present invention can effectively load TSA peptide MUC1 to ripe DC cell-targeting.
atgcaggctg tggtgactca ggagagtgcc ctgaccacat cacctggcga gacagtgact 60
ctgacctgtc ggagctccac cggggcagtg acaatcagca actacgccaa ttgggtccag 120
gaaaaaccag accacctgtt cacagggctg attggcggga ctaacaatcg agcacccgga 180
gtgcctgcca ggtttagtgg gtcactgatc ggagacaagg ccgctctgac aattactggc 240
gcccagaccg aggatgaagc tatctacttc tgtgcactgt ggtataacaa tcagttcatt 300
tttgggagcg gaacaaaagt gactgtcctg ggcggcggag gaggaagcgg aggaggaggg 360
tccggaggcg ggggatctgg aggaggagga agtgaggtgc agctgcagca gtctggccct 420
gtgctggtca agccaggggc tagtgtgaaa atgtcatgca aggcaagcgg caacaccttc 480
acagacagct ttatgcactg gatgaaacag tcccatggaa agtctctgga gtggatcggc 540
atcattaacc cctacaatgg gggaacctcc tataatcaga agttcaaagg caaggccact 600
ctgaccgtgg ataaatctag ttcaaccgct tacatggagc tgaacagcct gacatccgaa 660
gactctgccg tgtactattg tgctcgcaat ggcgtccgat actattttga ttattggggc 720
caggggacta ccctgacagt gagcacagca cctccagctc atggtgtgac tagcgctcca 780
gatacccgcc cggcacccgg gtctacggcg cctccggaaa acctgtattt tcagggcgaa 840
cagaagctga ttagcgaaga ggatctgcac catcatcacc accat
Claims (4)
1. method that loads tumor antigen peptide to dendritic cell DC target, it is characterized in that: utilize gene vitro recombination and the expression of antibody gene and the codes for tumor antigen peptide MUC1 of anti-DEC-205 molecule, the fusion rotein V of acquisition and the efficient combination of dendritic cell DC surface DEC-205 acceptor molecule, obtain the tumor antigen peptide MUC1 that the dendritic cell target is loaded.
2. to dendritic cell DC target, load according to claim 1 the method for tumor antigen peptide, it is characterized in that comprising the following steps:
1) will resist the antibody gene of DEC-205 and the gene of codes for tumor antigen peptide MUC1 synthesize and recombinate external, obtaining preserving number CGMCC is the expression plasmid pET41a-antiDEC-ScFV of NO:7686;
2) expression plasmid pET41a-antiDEC-ScFV is proceeded to intestinal bacteria, through inducing, obtain expressed fusion protein V precursor;
3) by fusion rotein V precursor after denature and renature, obtain the fusion rotein V with active function;
4) adopt biotin labeled fusion rotein V with active function to be combined with dendritic cell DC, with the fluorescently-labeled affine tumor antigen peptide MUC1 that the dendritic cell target is loaded that usually detects.
3. to dendritic cell DC target, load according to claim 2 the method for tumor antigen peptide, it is characterized in that in described step 1), the antibody molecule of anti-DEC-205 and tumor antigen peptide MUC1 are binned in same fusion rotein.
4. the method that loads tumor antigen peptide to dendritic cell DC target according to claim 2, it is characterized in that in described step 2) in, the nucleotide sequence that the genosome of the antibody gene of anti-DEC-205 molecule and codes for tumor antigen peptide MUC1 is reassembled into outward, be cloned on protein expressing plasmid, through inducing at expression in escherichia coli.
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| CN110904050A (en) * | 2019-12-16 | 2020-03-24 | 启辰生生物科技(珠海)有限公司 | Engineered antigen presenting cells, immunomodulatory compositions, and uses |
| CN110904050B (en) * | 2019-12-16 | 2021-02-05 | 启辰生生物科技(珠海)有限公司 | Engineered antigen presenting cells, immunomodulatory compositions, and uses |
| CN115028741A (en) * | 2022-06-21 | 2022-09-09 | 苏州工业园区唯可达生物科技有限公司 | Tumor antigen-antibody complex, preparation method and application thereof |
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