CN103981191A - Plant seed expression system for single-chain antibody against CD20 - Google Patents
Plant seed expression system for single-chain antibody against CD20 Download PDFInfo
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Abstract
The invention discloses a plant seed expression system for a single-chain antibody against CD20. According to the invention, a single-chain antibody gene against CD20 is designed and artificially synthesized according to the sequence of VL-VH-Linker-hIgG1Fc by adding the nucleotide sequence of a signal peptide sequence coding barley alpha-Amylase at the 5' end of the gene, the gene is inserted into a plant seed specific expression vector p1301b, and an Agrobacterium-mediated method is employed for transformation of plants; an expression frame with a seed specific promoter, the single-chain antibody gene against CD20 and a terminator is integrated into the genomes of the plants; plants with the gene are screened and cultured, screening and propagation are further carried out so as to obtain a homozygous plant, and seeds produced by the homozygous plant contain the single-chain antibody against CD20; recombinant protein is directionally expressed in vacuole. The expressed single-chain antibody against CD20 has biological activity, and ADCC killing capability of the antibody on Daudi cells is not significantly different from that of Rituxan.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to anti-CD20 single-chain antibody gene and utilize the method for plant seed specific expressing promoter anti-CD20 single-chain antibody of high efficient expression in plant seed.
Background technology
According to the Chinese tumour registration of < < 2012 annual report > >, show, the annual new cases of cancer approximately 3,120,000 of current China, because cancer mortality surpasses 2,000,000.Malignant tumour has become first cause of death of our urban and rural residents.Non-Hodgkin′s lymphomas (Non Hodgkin ' s lymphoma, NHL) is modal lymphsystem malignant tumour, and wherein the overwhelming majority is B cell lymphoma.CD20 antigen is a kind of B cell differentiation antigen, is positioned at the surface of B cell, exists only in mature B cell and B cell precursor, does not express, and all have overexpression in healthy tissues in more than 95% B cell lymphoma.This also makes CD20 become a desirable target spot for the treatment of B cell lymphoma.Rituxan(Rituximab, Mabthera) be U.S. Roche Holding Ag product, be a kind of human mouse chimeric antibody.Rituxan obtained FDA approval listing in 1997, be first monoclonal antibody that is approved for treatment malignant tumour.Be used for the treatment of at present low evil degree, follicular B cell lymphoma.
Along with Rituxan being treated to going deep into of some other autoimmune disorder research, the potential value of Rituxan aspect clinical disease treatment will further be developed, and society is also increasing to its demand.But the Rituxan being produced by mammalian cell (CHO) needs expensive equipment and substratum, thereby has increased its production cost, causes its price high (approximately 200,000 yuans of left and right of the expense of a course for the treatment of).
Utilize plant-bioreactor successfully to express the multiple pharmaceutical protein with biological activity and function, comprising monoclonal antibody, vaccine antigen, enzyme, blood protein, cytokine, somatomedin and tethelin etc. for treatment.The plant of usining has many features and advantage as bio-reactor: (1) can select multi-form expression system according to the demand of recombinant protein, can be divided into whole strain expression and tissue specific expression, as seed, fibrous root, and suspension cell etc.; (2) there is lower production cost and handiness during scale operation; (3) plant has modification approach after the protein translation similar to mammalian cell, thereby obtains the activated protein that has function; (4) higher security, does not carry animal pathogen; (5) be convenient to storage and transportation.
Although the spermatophyte such as beans and cereal makes the less biomass of deposits yields with respect to leaf class, for the splendid condition that provides is provided.Leaf class crop is after sampling, and blade needs freezing or dry in order to transportation, or processes immediately; And ripe seed can at room temperature be stored for a long time.In long-term evolutionary process, seed, as the storage organ of plant, can accumulate a large amount of protein in very little space, and in seed, contains the folding environment of abundant molecular chaperones, adequate proteins matter and less protein kinase.In addition, most seed small volume, can make the recombinant protein high concentration expressed like this, although that total biological yield does not have blade plant is high, is conducive to downstream extraction and the purifying of protein; And plant seed expression recombinant protein, can not affect the growth of plant.And by recombinant protein orientation expression is stored to vacuole, not only can obtain complete posttranslational modification, can also improve the expression amount of recombinant protein.
Summary of the invention
The object of this invention is to provide a kind of fusion gene, anti-CD20 single-chain antibody gene, and utilize plant seed expression system to express anti-CD20 single-chain antibody gene, produce anti-CD20 single-chain antibody.
Anti-CD20 single-chain antibody gene, it comprises: the Fc fragment of variable region of light chain-variable region of heavy chain-connection peptides-immunoglobulin G while and added the signal peptide sequence of coding barley α-Amylase at its 5 ' end;
Described anti-CD20 single-chain antibody gene, its base sequence is as shown in SEQ ID NO.1;
A plant expression carrier plasmid, it is in plant expression vector, to have inserted above-mentioned anti-CD20 single-chain antibody gene;
Described plant expression vector is plant seed specific expression carrier p1301b;
Described plant seed specific expression carrier p1301b, by following method, built: synthetic promotor and the terminator sequence of β-phaseolin, and at promotor two ends, add respectively Xho I and Nco I restriction enzyme site, at terminator two ends, add respectively Hind III and Kpn I restriction enzyme site; And from pBASTA plasmid, obtain 35S+Bar gene by the method for KpnI and Bst EII double digestion; Utilize Kpn I and Bst EII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas.By enzyme, cut, ligation is connected to the promotor of β-phaseolin and terminator original paper in p1301Bas again;
Anti-CD20 single-chain antibody, it is produced by following method:
1,, by above-mentioned a kind of plant expression carrier plasmid, be converted in agrobacterium tumefaciens EHA105;
2, again by agrobacterium tumefaciens transfection to plant;
3, cultivate described plant, gather seed; Extract anti-CD20 single chain antibody protein;
Described plant is Arabidopis thaliana, safflower, corn or butch flax.
The application of above-mentioned anti-CD20 single-chain antibody in preparation treatment non-Hodgkin′s lymphomas and autoimmune disorder medicine.
The invention provides a kind of plant seed expression system of anti-CD20 single-chain antibody, it is by VL-VH-Linker-hIgG1Fc order, the nucleotide sequence that has coding barley α-Amylase signal peptide sequence at 5 ' end of gene, design and synthetic anti-CD20 single-chain antibody gene, gene is inserted in plant seed specific expression carrier p1301b, by agrobacterium-mediated transformation transformed plant; Will be with seed specific promoters, the expression cassette of anti-CD20 single-chain antibody gene and terminator is integrated in Plant Genome; Screen and turned out the plant with said gene, further by screening and expanding numerous homozygous plants that obtained, in the seed that this homozygous plants produces, containing described anti-CD20 single-chain antibody; Recombinant protein orientation expression is in vacuole; And the anti-CD20 single-chain antibody of expressing has biologic activity, to the ADCC kill capability of Daudi cell and Rituxan without significant difference.
Adopting method of the present invention to produce anti-CD20 single-chain antibody tool has the following advantages:
1,, by adopting secretion type expression, recombinant protein can obtain correct folding, assembling and modifying, and orientation is stored in vacuole, guaranteed the acquisition of the biologic activity of restructuring pharmaceutical protein;
2, utilize expression of plants foreign protein can avoid the pollution of escherichia coli endotoxin and animal pathogen;
3, by utilizing seed-specific strong promoter, can effectively improve the expression amount of recombinant protein;
4, utilize plant seed expression system to produce anti-CD20 single-chain antibody, can effectively reduce production costs, be conducive to the industrialization of anti-CD20 single-chain antibody simultaneously.
Accompanying drawing explanation
Fig. 1 is that the enzyme of anti-humen CD 20 single-chain antibody gene of the vegetable codon Preference of synthetic is cut evaluation figure; Wherein: 1, DL2000,2--4, plasmid, 5, control plasmid, 6, DL15000;
Fig. 2 is the building process schematic diagram of plant seed expression vector p1301-Ri;
Fig. 3 is the double digestion evaluation figure of expression vector p1301-Ri; Wherein: 1, DL2000,2, plasmid;
Fig. 4 is the structure detailed maps of expression vector p1301-Ri;
Fig. 5 is the SDS-PAGE electrophorogram of transgenic arabidopsis seed; Wherein: 1, non-transgenic Arabidopis thaliana, 2--4, transgenic arabidopsis;
Fig. 6 is the Western Blot detection figure that anti-humen CD 20 single-chain antibody is expressed in Arabidopis thaliana seed; Wherein: 1--6, transgenic arabidopsis, 7, non-transgenic Arabidopis thaliana;
Fig. 7 is the biologic activity detection figure of anti-humen CD 20 single-chain antibody.
Embodiment
the acquisition of embodiment 1 intermediate carrier p1301b
According to TC.Hall and McBride, the nucleotide sequence that people's articles such as Summerfelt are announced, synthetic promotor and the terminator sequence of β-phaseolin, and at promotor two ends, add respectively Xho I and Nco I restriction enzyme site, at terminator two ends, add respectively Hind III and Kpn I restriction enzyme site; And from pBASTA plasmid, obtain 35S+Bar gene by the method for KpnI and Bst EII double digestion; Utilize Kpn I and Bst EII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas.By enzyme, cut, ligation is connected to the promotor of β-phaseolin and terminator original paper in p1301Bas, is built into intermediate carrier p1301b again.
the anti-CD20 single-chain antibody of embodiment 2, the i.e. synthetic of VL-VH-Linker-hIgG1Fc
The gene order of favorite plant codon of having entrusted Jin Weizhi bio tech ltd, Jiangsu synthetic, the encode nucleotide sequence of barley α-Amylase signal peptide sequence, the variable region of light chain of monoclonal antibody C2B8, connection peptides, the variable region of heavy chain of monoclonal antibody C2B8, human IgG1's Hinge and CH2 and CH3 sequence.In addition, at the two ends of composition sequence, add respectively Nco I and Hind III restriction enzyme site.Its base sequence is as shown in SEQ ID NO.1.
the structure of the anti-CD20 single-chain antibody of embodiment 3 seed expression vector
With Nco I and Hind III double digestion p1301b and the plasmid pUC57-Ri that contains above-mentioned single-chain antibody gene respectively, the fragment of the approximately 12000bp obtaining and 1500bp is connected, obtain the plant expression vector pAmy-scFv-Fc of anti-CD20 single-chain antibody.
the transformation of Arabidopsis thaliana that embodiment 4 is agriculture bacillus mediated and screening
1, Agrobacterium-mediated Transformation
Utilize freeze-thaw method that above-mentioned expression vector pAmy-scFv-Fc is proceeded in agrobacterium tumefaciens bacterial strain EHA105.7ul plasmid is added in 100ul Agrobacterium EHA105 competent cell and mixed gently, ice bath 30 minutes; Be placed in liquid nitrogen quick-frozen 2 minutes, be then placed in rapidly 37 ℃ of water-bath incubations 5 minutes.The YEP that adds 1mL antibiotic-free, under 28 ℃ of conditions, 180rpm recovery is 2 hours; After recovery, get 300ul bacterium liquid and be uniformly coated on the flat board that contains kantlex 50mg/L and Rifampin 25mg/L, be inverted for 28 ℃ and cultivate 2 days.Select independent colony inoculation in the YEP liquid nutrient medium that contains kantlex 50mg/L and Rifampin 25mg/L, 28 ℃ of incubated overnight;
Get the template that 2ul bacterium liquid detects as bacterium liquid PCR, reaction conditions is: 94 ℃ 5 minutes, (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 1 minute) totally 30 circulations, 72 ℃ 10 minutes, 4 ℃ 5 minutes.After reaction finishes, getting 20ul product detects with 1% agarose gel electrophoresis.
2, the conversion of Arabidopis thaliana and screening
By contaminating inflorescence method (Floral-Dip) arabidopsis thaliana transformation.
By the above-mentioned Agrobacterium that contains object expression vector, in 28 ℃ of overnight incubation, to OD600 ≈ 2.00,4000rpm collects thalline for centrifugal 10 minutes; Utilize freshly prepared resuspended liquid (5% sucrose, Selwet-77 300uL/L) that bacterial concentration is adjusted to OD600 ≈ 0.8.Get growth about month, upgrowth situation good stand, removes after the pod having grown, utilize dip-dyeing solution to contaminate over-ground part 30s, remove after unnecessary dip-dyeing solution with preservative film parcel, lucifuge is placed 48 hours, be placed under normal condition and grow, until seed maturity.
T1 collects after for seed fully matured and dries in the shade, and the seed after sterilization is evenly seeded on the MS substratum that contains careless ammonium phosphine 10mg/L, is placed in 4 ℃ of purifying and moves to illumination box after 48 hours, at 22 ℃, under the dark condition in 16 light/8, grows one week; Choose resistance seedling and move into continuation cultivation in soil.After seedling grows up, utilize blade to extract genomic dna, the seed that carries out collecting positive plant after PCR checking is for further analysis.
The PCR of embodiment 5 transgenic arabidopsis detects
The genomic dna of different individual plants of above-mentioned acquisition of take is template, utilizes respectively the primer that is positioned at strand gene two ends to carry out PCR detection, and reaction conditions is: 94 ℃ 5 minutes, (94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 1 minute) totally 30 circulations, 72 ℃ 10 minutes, 4 ℃ 5 minutes.After reaction finishes, getting 20ul product detects with 1% agarose gel electrophoresis.After reaction finishes, get PCR product and detect in 1% agarose gel electrophoresis, obtain the band of the approximately 1500bp of expection.
The extraction of embodiment 6 Arabidopis thaliana seed proteins
Look into and get 50 Arabidopis thaliana seeds and be placed in centrifuge tube, with 50ulTris-HCl(pH 8.0) grind, add the buffer as 10ul5X albumen loading after grinding fully, in boiling water, boil in 12000g centrifugal 30 minutes 10 minutes.Get 15ul supernatant and carry out electrophoresis.The results are shown in Figure 5, first is non-transgenic Arabidopis thaliana seed protein, and second Zhi Si road is transgenic arabidopsis seed protein, and haircut is depicted as recombinant protein.
In embodiment 7 Arabidopis thaliana seeds, the Western Blot of recombinant protein detects.
1, SDS-PAGE electrophoresis and the transferring film of seed protein
Seed protein carries out separation with 10% discontinuous Tricine-SDS-polyacrylamide gel in the vertical electrophoresis device of Bio-rad company, and condition is that 60V electrophoresis changes 100V into the tetrabromophenol sulfonphthalein gel of dishing out after 45 minutes.Electrophoresis finishes the protein electrophoresis transfer equipment of the rear Bio-rad of utilization company, and by protein delivery, to 0.45umPVDF film, condition is 100V electrophoresis 1 hour.
2, the hybridization of film
The film having shifted is first with the TBST confining liquid sealing that contains 5%BSA 2 hours, antibody is selected the anti-human IgG(H+L of HRP lotus root connection), to hatch after 1:1500 dilution, after cleaning, use the Super Signal west pico Chemiluminescent Substrate of Pierce to develop the color.Western Blot result shows in transgenic arabidopsis seed, there is the expression of anti-humen CD 20 single-chain antibody, and product size is about 53kD, consistent with expection.Western Blot analytical results is shown in Fig. 6.
the purifying of embodiment 7 recombinant fragment antibody
1, the extraction of seed total soluble protein
Take 500mg transgenic arabidopsis seed, after liquid nitrogen grinding is abundant, add 50ml hexane whirlpool concussion 10 minutes, rear centrifugal 5000g, 30 minutes, the cryodrying of supernatant discarded final vacuum.By dried powder liquid nitrogen grinding again, hexane extraction, abandons supernatant, vacuum dehydrating at lower temperature.With the resuspended precipitation of 50mM phosphate buffer solution (pH7.4) 45mL, ice bath 30 minutes.
By centrifugal 30 minutes of the good extracting solution 20000g of ice bath, carefully draw in the clean centrifuge tube of supernatant to, first through 0.45 μ m suction filtration, after recycling 0.22 μ m filter membrane suction filtration, obtain protein extract.
2, Protein A affinitive layer purification recombinant fragment antibody
By prepacked column HiTrap rProtein A FF(GE) be connected to AKTA purification system, with 50mM phosphate buffer solution, carry out after balance, by the protein extract upper prop after 0.22 μ m suction filtration, through 50mM phosphate buffer solution, wash away foreign protein, the rear 0.1M of utilization citrate buffer solution carries out wash-out recombinant fragment antibody.(note: add in advance the 1MTris-HCl damping fluid of 100 μ LpH9.0 in collection tube, every pipe is collected 1ml elutriant)
the biologic activity of the anti-humen CD 20 single-chain antibody that embodiment 8 Arabidopis thalianas are expressed detects
1, the separation of peripheral blood mononuclear cell (PBMC)
Aseptic collection venous blood, injects the centrifuge tube that contains heparin 20U/ml, mixes gently, adds isopyknic PBS solution; Every pipe adds 6ml balance to the lymphocyte separation medium of room temperature, and inclination centrifuge tube slowly adds anticoagulation cirumferential blood 6ml/ pipe along tube wall; 20 ℃, centrifugal 30 minutes of 800g, natural reduction of speed; With suction pipe, insert gently ground-glass-like layer, slowly sucking-off peripheral blood mononuclear cell, puts into another 15ml centrifuge tube; After PBS dilution, adding without phenol red RPMI-1640 nutrient solution adjustment cell density is 6x10
6/ ml, adds the IL-2 preactivate of 160U/ml, is placed in 37 ℃, 5%CO
2standby in cell culture incubator.
2, the effect of cell and antibody
Take Daudi as target cell, and the phase cell of taking the logarithm respectively, uses without phenol red RPMI-1640 re-suspended cell to 3 * 10
5/ ml is standby; Set the maximum releasing value of substratum experimental port, background hole, effector cell+target cell spontaneous releasing value hole and effector cell+target cell hole, every kind of situation is all established 3 parallel holes; Restructuring scFv-Fc and Rituxan are diluted to concentration and are respectively 100 μ g/mL, 20 μ g/mL, 4 μ g/mL, 0.8 μ g/mL and 0.16 μ g/mL; According to the explanation of CytoTox 96 (Progema, USA) heterotope method cell killing detection kit, at 490nm, carry out the mensuration of light absorption value.Anti-humen CD 20 single-chain antibody has biologic activity, to the ADCC kill capability of Daudi cell and Rituxan without significant difference.See Fig. 7.
<110> Jilin Agriculture University
The plant seed expression system of a <120> anti-CD20 single-chain antibody
<160> 1
<210> 1
<211> 1494
<212> DNA
<213> is artificial
<400> 1
ccatggcgaa caaacacttg tccctctccc tcttcctcgt cctccttggc ctgtcggcca 60
gcttggcctc cgggcaaatt gttctctccc agtctccagc aatcctgtca gcttctcctg 120
gagagaaggt gactatgact tgcagggctt cttcatctgt ttcttacatc cactggttcc 180
agcagaagcc tggttcttca cctaagcctt ggatctacgc tacatctaac ttggcatctg 240
gagtgcctgt gaggttctct ggttctggtt caggtacttc ttactctttg acaatctcta 300
gggtggaggc tgaggacgct gctacttact actgccagca gtggacatct aaccctccaa 360
cattcggagg tggtactaag ttggagatca agggtggagg tggatctgga ggaggaggat 420
ctggtggagg aggttctcag gtgcagttgc aacagcctgg agctgagttg gtgaagcctg 480
gtgcttctgt gaagatgtct tgtaaggctt ctggatacac attcacttct tacaacatgc 540
actgggtgaa gcagactcct ggtaggggtt tggagtggat cggagctatc tacccaggaa 600
acggagacac atcttacaac cagaagttca agggtaaggc tacattgact gctgacaagt 660
cttcatctac tgcttacatg caattgtctt ctttgacatc tgaggactct gcagtttact 720
actgcgctag gtctacatac tacggaggtg actggtactt caacgtgtgg ggagcaggta 780
ccacggtcac tgtctctgca tctagagaca agactcacac ttgccctcca tgcccagctc 840
cagagttgtt gggaggacca tctgtgttcc ttttcccacc aaagccaaaa gacactctta 900
tgatctctag gactcctgag gtgacatgcg tggttgtgga tgtgtcacac gaggatcctg 960
aggtgaagtt caattggtac gtggacggtg tggaggtgca taacgcaaag actaagccaa 1020
gggaggagca gtacaactca acttacaggg tggtgtctgt gcttacagtg cttcaccagg 1080
actggttgaa tggtaaggaa tacaaatgta aggtgtcaaa caaggcattg cctgcaccta 1140
tcgagaagac tatttcaaag gcaaagggtc aaccaaggga gcctcaggtg tacactttgc 1200
caccttcaag ggaagagatg actaagaacc aagtttcttt gacttgcttg gtgaagggat 1260
tctacccttc tgacatcgca gtggaatggg agtctaacgg tcagccagaa aataactaca 1320
aaacaacacc tccagtgttg gactctgatg gttctttctt tttgtattca aagttgactg 1380
tggacaagtc aaggtggcag cagggtaacg tgttctcttg ctctgtgatg cacgaggcac 1440
ttcataacca ctacacacaa aagtctttgt cattgtcacc tggtaaataa gctt 1494
Claims (8)
1. anti-CD20 single-chain antibody gene, it comprises: the Fc fragment of variable region of light chain-variable region of heavy chain-connection peptides-immunoglobulin G while, and at its 5 ' end, added the signal peptide sequence of coding barley α-Amylase.
2. anti-CD20 single-chain antibody gene according to claim 1, is characterized in that: its base sequence is as shown in SEQ ID NO.1.
3. a plant expression carrier plasmid, it is in plant expression vector, to have inserted anti-CD20 single-chain antibody gene claimed in claim 1.
4. a kind of plant expression carrier plasmid according to claim 3, is characterized in that: described plant expression vector is plant seed specific expression carrier p1301b.
5. a kind of plant expression carrier plasmid according to claim 4, it is characterized in that: described plant seed specific expression carrier p1301b, by following method, built: synthetic promotor and the terminator sequence of β-phaseolin, and at promotor two ends, add respectively Xho I and Nco I restriction enzyme site, at terminator two ends, add respectively Hind III and Kpn I restriction enzyme site; And from pBASTA plasmid, obtain 35S+Bar gene by the method for KpnI and Bst EII double digestion; Utilize Kpn I and Bst EII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas,
By enzyme, cut, ligation is connected to the promotor of β-phaseolin and terminator original paper in p1301Bas again.
6. anti-CD20 single-chain antibody, it is produced by following method:
1), by above-mentioned a kind of plant expression carrier plasmid, be converted in agrobacterium tumefaciens EHA105;
2) again by agrobacterium tumefaciens transfection to plant;
3) cultivate described plant, gather seed, extract anti-CD20 single chain antibody protein.
7. anti-CD20 single-chain antibody according to claim 1, is characterized in that: described plant is Arabidopis thaliana, safflower, corn or butch flax.
8. the application of anti-CD20 single-chain antibody claimed in claim 6 in preparation treatment non-Hodgkin′s lymphomas and autoimmune disorder medicine.
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| CN106432497A (en) * | 2016-11-05 | 2017-02-22 | 吉林农业大学 | Method of using safflower carthamus suspension cells for producing CD20 antibody |
| CN109485704A (en) * | 2018-11-27 | 2019-03-19 | 温州大学 | A kind of expression system of meningococcus fHbp albumen |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106432497B (en) * | 2016-11-05 | 2019-05-17 | 吉林农业大学 | Utilize the method for safflower suspension cell production CD20 antibody |
| CN109485704A (en) * | 2018-11-27 | 2019-03-19 | 温州大学 | A kind of expression system of meningococcus fHbp albumen |
| CN109485704B (en) * | 2018-11-27 | 2022-04-19 | 温州大学 | Expression system of meningococcal fHbp protein |
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| CN103981191B (en) | 2016-03-30 |
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