CN103981191B - A kind of plant seed expression system of anti-CD20 single-chain antibody - Google Patents
A kind of plant seed expression system of anti-CD20 single-chain antibody Download PDFInfo
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- CN103981191B CN103981191B CN201410142022.1A CN201410142022A CN103981191B CN 103981191 B CN103981191 B CN 103981191B CN 201410142022 A CN201410142022 A CN 201410142022A CN 103981191 B CN103981191 B CN 103981191B
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Abstract
The invention discloses a kind of plant seed expression system of anti-CD20 single-chain antibody, it is by VL-VH-Linker-hIgG1Fc order, the nucleotide sequence of encoding barley α-Amylase signal peptide sequence is had at 5 ' end of gene, design the anti-CD20 single-chain antibody gene of synthetic, gene is inserted in plant seed specific expression carrier p1301b, by Agrobacterium-mediated transformation plant; Will with seed specific promoters, the expression cassette of anti-CD20 single-chain antibody gene and terminator is integrated in Plant Genome; Screening and the plant turned out with said gene, further by screening with expand and numerously obtain homozygous plants, containing described anti-CD20 single-chain antibody in the seed that this homozygous plants produces; Recombinant protein orientation expression is in vacuole; The anti-CD20 single-chain antibody of expressing has biologic activity, to the ADCC kill capability of Daudi cell and Rituxan without significant difference.
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 the anti-CD20 single-chain antibody of plant seed specific expressing promoter high expression in plant seed.
Background technology
According to " 2012 Chinese tumour registration annual report " display, the annual new cancer cases of current China about 3,120,000, because cancer mortality is more than 2,000,000.Malignant tumour has become first cause of death of our urban and rural residents.Non Hodgkin lymphom (NonHodgkin ' slymphoma, NHL) be modal lymphoid malignancies, 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 in the normal tissue, and all has overexpression in B cell lymphoma more than 95%.This also makes CD20 become a promising target for the treatment of B cell lymphoma.Rituxan(Rituximab, Mabthera) be Roche Holding Ag of U.S. product, be a kind of human mouse chimeric antibody.Rituxan obtained FDA approval listing in 1997, be first monoclonal antibody being approved for treatment malignant tumour.Be used for the treatment of low evil degree, follicular B cell lymphoma at present.
Treat going deep into of some other autoimmune disorder research along with to Rituxan, the potential value of Rituxan in clinical disease treatment will be developed further, and society is also increasing to its demand.But the Rituxan produced by mammalian cell (CHO) needs expensive equipment and substratum, thus adds its production cost, causes its price high (expense of a course for the treatment of about about 200,000 yuans).
Plant-bioreactor is utilized successfully to have expressed the multiple pharmaceutical protein with biological activity and function, comprising monoclonal antibody, vaccine antigen, treatment enzyme, blood protein, cytokine, somatomedin and tethelin etc.As bio-reactor, then there is many features and advantage using plant: (1) can select multi-form expression system according to the demand of recombinant protein, whole strain expression and tissue specific expression can be divided into, 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, thus obtains the activated protein having function; (4) higher security, does not carry animal pathogen; (5) storage and transport is convenient to.
Although the spermatophyte such as beans and cereal produces less biomass relative to leaf class crop, provide splendid condition for storing.After sampling, blade needs freezing or dry in order to transport leaf class crop, or processes immediately; And the seed of maturation can at room temperature store 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 containing abundant molecular chaperones, environment that adequate proteins matter folds and less protein kinase in seed.In addition, most seed small volume, can make the recombinant protein high concentration of expression like this, although not have blade plant high for total biological yield, is conducive to downstream extraction and the purifying of protein; And plant seed expresses recombinant protein, can not affect the growth of plant.And by storing in recombinant protein orientation expression to vacuole, not only can obtain complete posttranslational modification, the expression amount of recombinant protein can also be improved.
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 add the signal peptide sequence of encoding barley α-Amylase at its 5 ' end;
Described anti-CD20 single-chain antibody gene, its base sequence is as shown in SEQIDNO.1;
A kind of plant expression carrier plasmid, it is in plant expression vector, insert 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, built by following method: the promotor of synthetic β-phaseolin and terminator sequence, and add XhoI and NcoI restriction enzyme site respectively at promotor two ends, add HindIII and KpnI restriction enzyme site respectively at terminator two ends; And obtain 35S+Bar gene by the method for KpnI and BstEII double digestion from pBASTA plasmid; Utilize KpnI and BstEII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas.Cut by enzyme, the promotor of β-phaseolin and terminator original paper are connected in p1301Bas by ligation 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 in plant;
3, the plant described in cultivation, gathers 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 lymphom 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 of encoding barley α-Amylase signal peptide sequence is had at 5 ' end of gene, design the anti-CD20 single-chain antibody gene of synthetic, gene is inserted in plant seed specific expression carrier p1301b, by Agrobacterium-mediated transformation plant; Will with seed specific promoters, the expression cassette of anti-CD20 single-chain antibody gene and terminator is integrated in Plant Genome; Screening and the plant turned out with said gene, further by screening with expand and numerously obtain homozygous plants, containing described anti-CD20 single-chain antibody in the seed that this homozygous plants produces; 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.
Adopt method of the present invention to produce anti-CD20 single-chain antibody tool to have the following advantages:
1, by adopting secretion type expression, recombinant protein can obtain correct folding, assembling and modification, and orientation is stored in vacuole, ensure that the acquisition of the biologic activity of restructuring pharmaceutical protein;
2, expression of plants foreign protein is utilized can to avoid the pollution of escherichia coli endotoxin and animal pathogen;
3, by utilizing seed-specific strong promoter, the expression amount of recombinant protein can effectively be improved;
4, utilize plant seed expression system to produce anti-CD20 single-chain antibody, effectively can reduce production cost, be conducive to the industrialization of anti-CD20 single-chain antibody simultaneously.
Accompanying drawing explanation
Fig. 1 is that the enzyme of the anti-humen CD 20 single-chain antibody gene of the vegetable codon Preference of synthetic cuts qualification 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 qualification 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 WesternBlot 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, the promotor of synthetic β-phaseolin and terminator sequence, and add XhoI and NcoI restriction enzyme site respectively at promotor two ends, add HindIII and KpnI restriction enzyme site respectively at terminator two ends; And obtain 35S+Bar gene by the method for KpnI and BstEII double digestion from pBASTA plasmid; Utilize KpnI and BstEII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas.Cut by enzyme, the promotor of β-phaseolin and terminator original paper are connected in p1301Bas by ligation, are built into intermediate carrier p1301b again.
the anti-CD20 single-chain antibody of embodiment 2, i.e. the synthetic of VL-VH-Linker-hIgG1Fc
Entrust Jin Weizhi bio tech ltd, the Jiangsu synthetic gene order of favorite plant codon, the i.e. nucleotide sequence of encoding 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, Hinge and CH2 of human IgG1 and CH3 sequence.In addition, NcoI and HindIII restriction enzyme site is added respectively at the two ends of composition sequence.Its base sequence is as shown in SEQIDNO.1.
the structure of embodiment 3 anti-CD20 single-chain antibody seed expression vector
With NcoI and HindIII double digestion p1301b and the plasmid pUC57-Ri containing above-mentioned single-chain antibody gene respectively, the fragment of about 12000bp with 1500bp obtained is connected, obtains 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
Freeze-thaw method is utilized to be proceeded in agrobacterium tumefaciens bacterial strain EHA105 by above-mentioned expression vector pAmy-scFv-Fc.7ul plasmid is added in 100ul Agrobacterium EHA105 competent cell and mix gently, ice bath 30 minutes; Be placed in liquid nitrogen quick-frozen 2 minutes, be then placed in rapidly 37 DEG C of water-bath incubations 5 minutes.Add the YEP of 1mL antibiotic-free, under 28 DEG C of conditions, 180rpm recovers 2 hours; Get after recovery on flat board that 300ul bacterium liquid is uniformly coated on containing kantlex 50mg/L and Rifampin 25mg/L, be inverted cultivation 2 days for 28 DEG C.Select independent colony inoculation in the YEP liquid nutrient medium containing kantlex 50mg/L and Rifampin 25mg/L, 28 DEG C of incubated overnight;
Get the template that 2ul bacterium liquid detects as bacterium liquid PCR, reaction conditions is: 94 DEG C 5 minutes, (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute) totally 30 circulations, 72 DEG C 10 minutes, 4 DEG C 5 minutes.Get 20ul product 1% agarose gel electrophoresis after reaction terminates to detect.
2, the conversion of Arabidopis thaliana and screening
By contaminating inflorescence method (Floral-Dip) arabidopsis thaliana transformation.
By the above-mentioned Agrobacterium containing object expression vector in 28 DEG C of overnight incubation, within centrifugal 10 minutes, collect thalline to OD600 ≈ 2.00,4000rpm; Utilize freshly prepared re-suspension liquid (5% sucrose, Selwet-77300uL/L) that bacterial concentration is adjusted to OD600 ≈ 0.8.Get growth about one month, upgrowth situation good stand, after removing the pod grown, utilize dip-dyeing solution to contaminate over-ground part 30s, wrap up with preservative film after removing unnecessary dip-dyeing solution, lucifuge places 48 hours, be placed on grown under normal conditions, until seed maturity.
T1 dries in the shade for collecting after seed fully matured, is evenly seeded on the MS substratum containing careless ammonium phosphine 10mg/L by the seed after sterilization, is placed in 4 DEG C of purifying and moves to illumination box after 48 hours, at 22 DEG C, grows one week under the condition that 16 light/8 are dark; Choose resistance seedling and move into continuation cultivation in soil.After seedling grows up, utilize blade to extract genomic dna, collect the seed of positive plant after carrying out PCR checking for further analysis.
The PCR of embodiment 5 transgenic arabidopsis detects
With the genomic dna of the different individual plants of above-mentioned acquisition for template, utilize the primer being positioned at strand gene two ends to carry out PCR detection respectively, reaction conditions is: 94 DEG C 5 minutes, (94 DEG C 30 seconds, 56 DEG C 30 seconds, 72 DEG C 1 minute) totally 30 circulations, 72 DEG C 10 minutes, 4 DEG C 5 minutes.Get 20ul product 1% agarose gel electrophoresis after reaction terminates to detect.Get PCR primer after reaction terminates to detect in 1% agarose gel electrophoresis, obtain the band of the about 1500bp expected.
The extraction of embodiment 6 Arabidopis thaliana seed protein
Look into and get 50 Arabidopis thaliana seeds and be placed in centrifuge tube, with 50ulTris-HCl(pH8.0) grind, add as 10ul5X albumen loadingbuffer after grinding fully, in boiling water, boil 10 minutes, in 12000g centrifugal 30 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 to four road is transgenic arabidopsis seed protein, and haircut is depicted as recombinant protein.
In embodiment 7 Arabidopis thaliana seed, the WesternBlot of recombinant protein detects.
1, the SDS-PAGE electrophoresis of seed protein and transferring film
Seed protein 10% discontinuous Tricine-SDS-polyacrylamide gel is separated in the vertical electrophoresis device of Bio-rad company, and condition is that 60V electrophoresis changes 100V into and to dish out gel to tetrabromophenol sulfonphthalein after 45 minutes.Electrophoresis terminates the rear protein electrophoresis transfer equipment utilizing Bio-rad company, and by protein delivery on 0.45umPVDF film, condition is 100V electrophoresis 1 hour.
2, the hybridization of film
The film shifted first closes 2 hours with the TBST confining liquid containing 5%BSA, the anti-human igg (H+L) that antibody selects HRP lotus root to join, to hatch after 1:1500 dilution, after cleaning, the SuperSignalwestpicoChemiluminescentSubstrate of Pierce is used to develop the color.WesternBlot result shows, has the expression of anti-humen CD 20 single-chain antibody in transgenic arabidopsis seed, and product size is about 53kD, consistent with expection.WesternBlot 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, adds 50ml hexane whirlpool and shake 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.By the resuspended precipitation of 50mM phosphate buffer solution (pH7.4) 45mL, ice bath 30 minutes.
By centrifugal for extracting solution 20000g good for ice bath 30 minutes, in the clean centrifuge tube of careful absorption supernatant to, first through 0.45 μm of suction filtration, after recycling 0.22 μm of filter membrane suction filtration, obtain protein extract.
2, ProteinA affinitive layer purification recombinant fragment antibody
By prepacked column HiTraprProteinAFF(GE) be connected to AKTA purification system, after balancing with 50mM phosphate buffer solution, by the protein extract upper prop after 0.22 μm of suction filtration, wash away foreign protein through 50mM phosphate buffer solution, the rear 0.1M of utilization citrate buffer solution carries out wash-out recombinant fragment antibody.(note: the 1MTris-HCl damping fluid adding 100 μ LpH9.0 in collection tube in advance, often 1ml elutriant collected by pipe)
the biologic activity of the anti-humen CD 20 single-chain antibody that embodiment 8 Arabidopis thaliana is expressed detects
1, the separation of peripheral blood mononuclear cell (PBMC)
Aseptic collection venous blood, injects the centrifuge tube containing heparin 20U/ml, mixes gently, add isopyknic PBS solution; Often pipe adds the lymphocyte separation medium of 6ml balance to room temperature, and inclination centrifuge tube, slowly adds anticoagulation cirumferential blood 6ml/ along tube wall and manage; 20 DEG C, centrifugal 30 minutes of 800g, natural reduction of speed; Insert ground-glass-like layer gently with suction pipe, slow sucking-off peripheral blood mononuclear cell, put into another 15ml centrifuge tube; Adding without phenol red RPMI-1640 nutrient solution adjustment cell density after PBS dilution is 6x10
6/ ml, adds the IL-2 preactivate of 160U/ml, is placed in 37 DEG C, 5%CO
2for subsequent use in cell culture incubator.
2, the effect of cell and antibody
Take Daudi as target cell, phase cell of taking the logarithm respectively, with without phenol red RPMI-1640 re-suspended cell to 3 × 10
5/ ml is for subsequent use; Setting substratum experimental port, background hole, effector cell+target cell Spontaneous release values hole and the maximum releasing value hole of effector cell+target cell, often kind of situation all establishes 3 parallel holes; Restructuring scFv-Fc and Rituxan is diluted to concentration and is respectively 100 μ g/mL, 20 μ g/mL, 4 μ g/mL, 0.8 μ g/mL and 0.16 μ g/mL; Carry out the mensuration of light absorption value at 490nm according to the explanation of CytoTox96 (Progema, USA) heterotope method cell killing detection kit.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 the anti-CD20 single-chain antibody of <120> mono-kind
<160>1
<210>1
<211>1494
<212>DNA
<213> is artificial
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gagtgcctgtgaggttctctggttctggttcaggtacttcttactctttgacaatctcta300
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cattcggaggtggtactaagttggagatcaagggtggaggtggatctggaggaggaggat420
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cagagttgttgggaggaccatctgtgttccttttcccaccaaagccaaaagacactctta900
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ttcataaccactacacacaaaagtctttgtcattgtcacctggtaaataagctt1494
Claims (6)
1. anti-CD20 single-chain antibody gene, is characterized in that: its base sequence is as shown in SEQIDNO.1.
2. a plant expression carrier plasmid, it is in plant expression vector, insert anti-CD20 single-chain antibody gene according to claim 1.
3. a kind of plant expression carrier plasmid according to claim 2, it is characterized in that: described plant expression vector is plant seed specific expression carrier p1301b, it is built by following method: the promotor of synthetic β-phaseolin and terminator sequence, and add XhoI and NcoI restriction enzyme site respectively at promotor two ends, add HindIII and KpnI restriction enzyme site respectively at terminator two ends; And obtain 35S+Bar gene by the method for KpnI and BstEII double digestion from pBASTA plasmid; Utilize KpnI and BstEII double digestion pCambia1301 simultaneously, be connected into 35S+Bar original paper, build intermediate carrier p1301Bas, then cut by enzyme, the promotor of β-phaseolin and terminator original paper are connected in p1301Bas by ligation.
4. anti-CD20 single-chain antibody, it is produced by following method:
1) by a kind of plant expression carrier plasmid of claim 2, be converted in agrobacterium tumefaciens EHA105;
2) again by agrobacterium tumefaciens transfection in plant;
3) plant described in cultivation, gathers seed, extracts anti-CD20 single chain antibody protein.
5. anti-CD20 single-chain antibody according to claim 4, is characterized in that: step 2) described in plant be Arabidopis thaliana, safflower, corn or butch flax.
6. the application of anti-CD20 single-chain antibody according to claim 4 in preparation treatment non Hodgkin lymphom and autoimmune disorder medicine.
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| CN106220733A (en) * | 2016-08-17 | 2016-12-14 | 林志国 | A kind of single-chain antibody and application thereof |
| CN106432497B (en) * | 2016-11-05 | 2019-05-17 | 吉林农业大学 | Utilize the method for safflower suspension cell production CD20 antibody |
| CN109485704B (en) * | 2018-11-27 | 2022-04-19 | 温州大学 | Expression system of meningococcal fHbp protein |
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