CN109385445A - Plant cuts down the application in antibody in expression shellfish as host - Google Patents
Plant cuts down the application in antibody in expression shellfish as host Download PDFInfo
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- CN109385445A CN109385445A CN201710687275.0A CN201710687275A CN109385445A CN 109385445 A CN109385445 A CN 109385445A CN 201710687275 A CN201710687275 A CN 201710687275A CN 109385445 A CN109385445 A CN 109385445A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
- C12N15/8258—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon for the production of oral vaccines (antigens) or immunoglobulins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
Abstract
The present invention relates to field of biotechnology, in particular to the plant application of cutting down antibody in expression shellfish as host.The present invention is expressed shellfish using simple and effective mediated by agriculture bacillus vacuum infiltration methods and cuts down monoclonal antibody (Avastin, bevacizumab, Arastin) using plant such as romaine lettuce as the effective expression platform of recombinant protein production.The expression system determines that plant foreign protein can be collected after Agrobacterium infects 4d.Determine that recombination shellfish cuts down antibody successful expression using SDS-PAGE method.Breast cancer cell Inhibition test proves that the shellfish of romaine lettuce production cuts down antibody and has the biological activity for killing cancer cell.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant cuts down the application in antibody in expression shellfish as host.
Background technique
The main reason for cancer is dead all over the world, due to population growth and aging and other factors it is universal
In the presence of such as air, food etc. pollutes, and incidence is rising.In treatment method, operation, chemotherapy and radiation irradiation according to
It is so the main means for treating various tumor types and stage at this stage.However, due to lacking selectivity, chemotherapy to tumour cell
The success rate of method is limited, and will lead to general toxicity and drug resistance.Radiation therapy can not all kill cancer cell, and
Cause patient's sick body weaker.Because cancer cell has diffusivity, operation can cut off site of pathological change, but can not stop
The propagation of cancer cell.Therapy more advanced at present is the characterization of molecules according to tumour cell, designs better targeted therapy
It is prevented to grow and propagate.Most of in these therapies be all based on the small-molecule drug for being easily accessible tumour cell or and its
The monoclonal antibody (mAb) that particular target on surface combines.
Targeted therapy based on mAb is the immunotherapy for different target, such as blocks oncogenic pathways, then to cell
The influence of growth and apoptosis blocks neovascularization, adjusts to the immune response of tumour cell, the adjusting of osteoclast function or
Tumour cell is killed in the delivering of cytotoxic drug.Since food and drug administration (FDA) ratifies the first Dan Ke
Grand antibodySince, including mouse, hundreds of antibody including chimeric and humanized antibody have been developed that use
In treatment of cancer.Some in these monoclonal antibodies are ratified by FDA, and can be used for the clinic in everyday practice
Using, it is combined as monotherapy or with standard chemotherapy regimen, and many other monoclonal antibodies are still in different clinical tests
In tested.
It is a plurality of types of for treating that shellfish cuts down antibody (Avastin, bevacizumab, Arastin) bevacizumab drug
Cancer and specific ophthalmology disease.Treat colon cancer by being slowly injected into venous patient, lung cancer, glioblastoma, kidney is thin
Born of the same parents' cancer.Bevacizumab can also treat age-related macular degeneration by ocular injection.Bevacizumab is to belong to blood vessel life
At the drug of inhibitor and monoclonal antibody family.It inhibits the growth of tumour cell by slowing down the growth of new blood vessel.?
The U.S., medical bevacizumab approval was in 2004.Bevacizumab is also a kind of standing drug for treating eye illness.
Shellfish cut down antibody be the World Health Organization approval substantially standing drug, inside health system be also it is most effective and
Safe drugs.Monoclonal antibody mainly is cut down using zooblast production shellfish at this stage.But animal cell culture needs price
Expensive culture solution, stringent workshop condition is complicated for operation, and the time cycle at least two weeks, and also zooblast production capacity is low,
Cause cost high.Sometimes the virus of zooblast institute band can infect the mankind, cause safety low.
Summary of the invention
In view of this, the present invention provides plant as host expression shellfish cut down the application in antibody.The present invention utilizes plant
The efficient platform technology that object especially romaine lettuce is produced as recombinant protein, expresses shellfish and cuts down antibody.And under mild conditions
It is successfully separated out active foreign protein, it was demonstrated that plant especially romaine lettuce expression platform, which successfully can be used to produce shellfish, cuts down antibody
Albumen.Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, the potential pest and disease damage etc. of infection human body is eliminated.Greatly
It is big to reduce production cost, improve Product Safety.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides plant as host expression shellfish cut down the application in antibody.Preferably, the antibody is Dan Ke
Grand antibody.The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean or wheat；The organ of the plant be selected from seed, leaf, rhizome or
Whole plant.The present invention also provides a kind of expression vector, the sequence of heavy chain cut down including shellfish or sequence of light chain carrier:
In some specific embodiments of the invention, the sequence of heavy chain or sequence of light chain that the shellfish is cut down are that shellfish is cut down to weight
Chain, shellfish cut down light chain codon optimization be favorite plant codon, sequence of heavy chain that the shellfish of the optimization of acquisition is cut down or optimization
The sequence of light chain that shellfish is cut down.
In some specific embodiments of the invention, the sequence of heavy chain that the shellfish of the optimization is cut down such as SEQ ID No.1 institute
Show；The nucleotide sequence for the heavy chain that the shellfish of the optimization is cut down is as shown in SEQ ID No.2；
The sequence of light chain that the shellfish of the optimization is cut down is as shown in SEQ ID No.3；The nucleosides for the light chain that the shellfish of the optimization is cut down
Acid sequence is as shown in SEQ ID No.4.
In some specific embodiments of the invention, the carrier is binary plant carrier.
In some specific embodiments of the invention, the construction method of the expression vector includes the following steps:
Step 1: shellfish is cut down into heavy chain respectively, shellfish cut down light chain codon optimization be favorite plant codon, obtain:
I. the sequence of heavy chain that the shellfish optimized is cut down；
The sequence of light chain that the shellfish of II optimization is cut down；
Step 2: being separately added into 5 ' ends of the sequence of light chain that the shellfish of the shellfish of the optimization sequence of heavy chain cut down or optimization is cut down
Xbal restriction enzyme site is separately added into Sac I site in 3 ' ends；
It is grand into pUC57 carrier by golden Stryker, pBeva-H or pBeva-L cloning vector is obtained respectively；
Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 respectively by Xbal/Sacl, is cloned into double base
Plant vector pCam35S obtains expression vector p35S-Beva-H or p35S-Beva-L respectively.
Specifically, in order to provide high efficient expression of the foreign protein in plant, people shellfish is cut down heavy chain (GenBank by the present invention
No. Accession: AAO17823.1), light chain, the anti-translation software of (No. GenBankAccession: 4DKE_L) protein sequence
Its its codon optimization is the codon of favorite plant by (https: //www.idtdna.com/CodonOpt), by Jin Sirui
Company (Nanjing, China) synthesis.5 ' end of weight chain-ordering is cut down in shellfish and is separately added into Xbal restriction enzyme site, in 3 ' ends
It is separately added into the site Sacl.And it is grand into pUC57 carrier (Fig. 1) by golden Stryker, pBeva-H or pBeva-L is obtained respectively
Cloning vector.Genetic fragment is separated from cloning vector respectively by XbaI/Sacl, and is cloned into binary plant carrier
PCam35S generates plant expression vector p35S-Beva-H and p35S-Beva-L respectively.
The present invention also provides the expression vectors to cut down the application in antibody in expression shellfish.
In addition, the present invention is provided altogether the present invention also provides a kind of method that plant cuts down antibody as host expresses shellfish
Expression vector be transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing protein,
It obtains shellfish and cuts down antibody.
Specifically, Four Plants expression vector p35S-Beva-H and p35S-Beva-L are passed through use respectively
Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.By institute
Bacterial strain is obtained equably to spread on the selective LB plate of antibiotic containing kanamycin (50mg/L).28 DEG C of incubations in the dark
After 2d, picking single colonie is inoculated into 0.5L YEB, and (yeast extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast are mentioned
Take object, 0.24g/L MgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation
Object is in oscillator (220rpm) with 25~28 DEG C of incubation 72h.OD600 value is measured by addition YEB culture medium and is adjusted to 3.5
~4.5.Then culture solution is collected, (4500 revolving speed) 10min is centrifuged.Agrobatcerium cell is resuspended in osmotic medium (10mM
MES, 10mM MgSO4) in O.D.600 be 0.5.
The Agrobacterium equivalent containing p35S-Beva-H or p35S-Beva-L prepared is mixed to O.D.600 and is
0.5.Culture suspension is placed in 2L beaker, is placed in drier.The romaine lettuce that this laboratory is saved is inverted (core is upward)
And lightly rotate in bacterial suspension, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is beaten
It opens to evacuate, and visual penetration liquid is in leaf tissue.Keep 30~60s of pressure state.Open the system quickly to discharge
Pressure makes penetrating fluid penetrate into the space in tissue.The process repeats 2~3 times, until high-visible penetrating fluid is in romaine lettuce tissue
Diffusion is obvious.Then romaine lettuce tissue is gently taken out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into modeling
In the container for expecting film covering.The sample of processing is kept into 4d in the dark.
In some specific embodiments of the invention, the mediated by agriculture bacillus vacuum infiltration includes the following steps:
Step 1: vacuumizing 25~45s；
Step 2: keeping 30~60s of vacuum (- 95kPa) pressure；
Step 3: releasing stress so that penetrating fluid penetrates into the plant tissue；
It repeats the above steps 2~3 times, is protected from light processing 4d.
In some specific embodiments of the invention, Agrobacterium is specially Agrobacterium tumefaciens GV3101.
Clone pBeva-H of the present invention and pBeva-L genetic fragment (Fig. 2), and construct four kinds of binary plant expression
For carrier p35S-Beva-H or p35S-Beva-L after completing construct, being digested with specific restriction enzyme confirms that genetic fragment has been
Whole.After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, other than firm middle rib region, remaining part
Divide and shows khaki region after vacuum infiltration 4 days.
Extracting and developing protein specifically: the romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with blender, and uses body
Product is than Extraction buffer (100mM KPi, the pH7.8 for 1:1 ratio；5mM EDTA；10mM beta -mercaptoethanol) it is high in blender
1~2min of speed homogenate.Homogenate is adjusted to pH8.0, with filtered through gauze, filtrate 4 DEG C with 10,000g be centrifuged 15min with
Remove cell fragment.Supernatant is collected, is mixed with ammonium sulfate (50%), and shakes be incubated for 60min on ice.Pass through centrifuge
(10,000g) separates 15min at 4 DEG C again.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, on ice
Suspension 60min is shaken, 15min is centrifuged with 10,000g at 4 DEG C again.Then, liquid is discarded supernatant, sample pellet egg will be handled
White matter is dissolved in 5mL buffer (20mM KPi, pH 7.8；2mM EDTA；10m M beta -mercaptoethanol) in and store at 4 DEG C.
PAGE gel electrophoresis specifically: collect the protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce, sampling
(95 DEG C) load buffers (Biorad, Hercules, CA, USA) of product (5 μ L) thermal denaturation are in 4-12%Bis-Tris
Plus SDS- denaturant gel (ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, in non-change
Property gel electrophoresis in detect antibody affine degree.Then gel is carried out again after being dyed with Coomassie blue G250 (Biorad)
It takes pictures.
The Downstream processing of the recombinant protein of plant origin is typically difficult to and valuableness, because cellulose cell wall is difficult to split
Solution and secondary plant metabolites.We are stirred with blender and are homogenized, and greatly save homogenate cost and technique.Recombination shellfish is cut down
Antibody separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about 20 and 50kDa band in swimming lane
(Fig. 3 A) meets the albumen size that shellfish cuts down antibody light and weight chain.About 150kDa (figure is observed in non denatured gel electrophoresis
Band 3B), it was demonstrated that romaine lettuce recombination light and weight chain is successfully combined into antibody structure, meets the antibody protein molecular weight that shellfish is cut down.Base
Measuring the protein content of purification of samples in Bradford measuring method and spectrodensitometry control group is about 0.68mg/g.
The antibody for inhibiting verification experimental verification to obtain through cancer cell, human breast cancer cell line BT-474 cell are being supplemented with 10%
It is raw in the RPMI-1640 culture medium (Gibco, USA) of FBS (Gibco, USA), 100U/mL penicillin and 100 μ g/mL streptomysins
It is long.5%CO of all cells at 37 DEG C2It is cultivated in humidification atmosphere, updates culture medium daily, and every using 0.25% trypsase
Three days passage cells.A549 cell is collected by trypsin digestion, and with 1 × 106The density resuspension of a cell/mL, adds
The 10 μ g shellfishes for entering to be separately added into purifying cut down antibody, are used for after 84h with annexin V-fluorescein isothiocyanate (FITC) and PI
It is double to contaminate to assess the ratio of apoptotic cell.
The inhibition that purifying shellfish cuts down antibodies on breast cancer cell (BT-474) is studied by cell experiment.By the way that purifying is added
Recombination shellfish cuts down antibody culture BT-474 cell, checks that cell growth result shows without any processing at the time point of 84h
BT-474 cell well-grown.In contrast, largely (Fig. 4) is disappeared using the cell that the recombination shellfish of purifying cuts down antibody culture.
These results indicate that cutting down antibody with biological activity by the external source shellfish of romaine lettuce system Transient Expression and BT- can be killed
474 cells.The result shows that plant especially romaine lettuce is a kind of suitable bioreactor for producing shellfish and cutting down antibody.
The present invention cuts down antibody using romaine lettuce come transient expression shellfish, and (4d) can produce the albumen of high-content in a relatively short period of time
Matter.Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this side
Method reduces bio-safety problem to the maximum extent, because processed romaine lettuce tissue is usually in completely enclosed facility or appearance
It is developed in device, biological pollution problem is not present.Romaine lettuce is substantially free of plant noxious material, and itself fiber is few, is conducive to
The protein purification in downstream.Monoclonal antibody is cut down using romaine lettuce system production shellfish, production cycle and production cost can be greatly shortened.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows cloning vector pUC57 schematic diagram；
Fig. 2 shows that shellfish cuts down plant binary expression vector p35S-Beva-H (heavy chain) and p35S-Beva-L (light chain) building stream
Journey；Using restriction enzyme (Xbal/SacI) double digestion, cut that shellfish cuts down H and shellfish cuts down L weight respectively from Fig. 1 cloning vector
Chain segment connects the site XbaI/SacI into pCam35S, generates plant binary expression vector p35S-Beva-H and p35S-
Beva-L；
LB and RB:Ti plasmid right boundary；35S, the CaMV 35S with tobacco mosaic virus (TMV) (TMV) 5 ' UTR are opened
Mover；NPT II, the expression of the coding nptII gene for kalamycin resistance；Nos 3 ', terminator；
Fig. 3 (A) shows PAGE gel electrophoresis result；Swimming lane 1: shellfish cuts down recombinant antibodies；
Fig. 3 (B) shows native gel electrophoresis result；Swimming lane 2: shellfish cuts down recombinant antibodies；
Fig. 4 shows that studying purifying shellfish by cell experiment cuts down antibody to mammary carcinoma cells (BT-474) inhibition.
Specific embodiment
The invention discloses plant as host expression shellfish cut down the application in antibody, those skilled in the art can use for reference
Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.Method of the invention and application by compared with
Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to as described herein
Methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, be
A kind of method of quick transient expression recombinant protein.The vacuum Agrobacterium permeating method that the present invention describes is simple, quickly, and
And recombinant protein yield can be improved.Romaine lettuce can increase protein output by bearing vacuum pressure, and allow every leaf
The more complete infiltration of son.Due to romaine lettuce be easy to grow and can commercial mass production, than other transient expression plants, such as
Tobacco etc. is easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can be significantly reduced.It is comprehensive
Upper described, the present invention can use large-scale production shellfish in the romaine lettuce system short time and cut down monoclonal antibody.
Plant provided by the invention cuts down in the application in antibody that raw materials used and reagent can be by as host in expression shellfish
Market is bought.
Below with reference to embodiment, the present invention is further explained:
The building of 1 plant transient expression vector of embodiment
In order to provide high efficient expression of the foreign protein in plant, by people shellfish cut down heavy chain (GenBank Accession:
AFR78283.2), light chain, (GenBank No. Accession: 1BJ1-L) anti-translation software of protein sequence (https: //
Www.idtdna.com/CodonOpt) by its its codon optimization be favorite plant codon, by Jin Sirui company (Nanjing,
China) synthesis.The end weight chain-ordering 5' is cut down in shellfish and is separately added into Xbal restriction enzyme site, is separately added into the end 3'
The site SacI.And (Fig. 1) is cloned into pUC57 carrier by Jin Sirui company, pBeva-H and pBeva-L clone is generated respectively to be carried
Body.Genetic fragment is separated from cloning vector respectively by Xbal/Sacl, and is cloned into binary plant carrier, and pCam35S divides
It Chan Sheng not plant expression vector p35S-Beva-H and p35S-Beva-L.Two kinds of plant expression vectors are passed through into use respectively
Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.By institute
Bacterial strain is obtained equably to spread on the selective LB plate of antibiotic containing kanamycin (50mg/L).28 DEG C of incubations in the dark
After 2d, picking single colonie is inoculated into 0.5L YEB, and (yeast extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast are mentioned
Take object, 0.24g/L MgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation
Object is in oscillator (220rpm) with 25~28 DEG C of incubation 72h.OD600 value is measured by addition YEB culture medium and is adjusted to 3.5
~4.5.Then culture solution is collected, (4500 revolving speed) 10min is centrifuged.Agrobatcerium cell is resuspended in osmotic medium (10mM
MES, 10mM MgSO4) in O.D.600 be 0.5.
Clone obtains pBeva-H and pBeva-L genetic fragment (Fig. 2), and constructs two kinds of binary plant expression vectors
P35S-Beva-H and p35S-Beva-L.After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete
's.After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, other than firm middle rib region, rest part
Khaki region is shown after vacuum infiltration 4 days.
The vacuum infiltration of 2 mediated by agriculture bacillus of embodiment
The Agrobacterium equivalent containing p35S-Beva-H and p35S-Beva-L prepared is mixed to O.D.600 and is
0.5.Culture suspension is placed in 2L beaker, is placed in drier.The romaine lettuce that this laboratory is saved is inverted (core is upward)
And lightly rotate in bacterial suspension, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is beaten
It opens to evacuate, and visual penetration liquid is in leaf tissue.It is kept for pressure state 30~60 seconds.Open the system quickly to discharge
Pressure makes penetrating fluid penetrate into the space in tissue.The process repeats 2 to 3 times, until high-visible penetrating fluid is in romaine lettuce tissue
Diffusion is obvious.Then romaine lettuce tissue is gently taken out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into modeling
In the container for expecting film covering.The sample of processing is kept 4 days in the dark.
3 Protein Extraction of embodiment and separation
Romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with blender, and is buffered with the extraction that volume ratio is 1:1 ratio
Liquid (100mM KPi, pH7.8；5mM EDTA；10m M beta -mercaptoethanol) homogenate of blender high speed 1-2 minutes.By homogenate
It is adjusted to pH 8.0, with filtered through gauze, filtrate is centrifuged 15 minutes with 10,000g at 4 DEG C to remove cell fragment.In collection
Clear liquid is mixed with ammonium sulfate (50%), and shakes be incubated for 60 minutes on ice.By centrifuge (10,000g) at 4 DEG C again
Separation 15 minutes.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, shakes suspend 60 minutes on ice, exist again
With 10,000g centrifugation 15 minutes at 4 DEG C.Then, liquid is discarded supernatant, processing sample pellet protein is dissolved in 5mL buffer
(20mM KPi, pH 7.8；2mM EDTA；10m M beta -mercaptoethanol) in and store at 4 DEG C.
The Downstream processing of the recombinant protein of plant origin is typically difficult to and valuableness, because cellulose cell wall is difficult to split
Solution and secondary plant metabolites.We are stirred with blender and are homogenized, and greatly save homogenate cost and technique.
Shellfish, which cuts down antibody and separates us by denaturant gel SDS-PAGE and observe in swimming lane, estimates that molecular weight is about 20
And 50kDa band (Fig. 3 A), meet the albumen size that shellfish cuts down antibody light and weight chain.It is observed in non denatured gel electrophoresis big
The band of about 150kDa (Fig. 3 B), it was demonstrated that romaine lettuce recombination light and weight chain is successfully combined into antibody structure, meets the antibody egg that shellfish is cut down
White molecular weight.Based on Bradford measuring method and spectrodensitometry control group measurement purification of samples protein content be about
0.68mg/g。
4 PAGE gel electrophoresis of embodiment
The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) of thermal denaturation of sample (5 μ L) loads are taken
Buffer (Biorad, Hercules, CA, USA) is 4~12%Bis-Tris Plus SDS- denaturant gel
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, it is detected in native gel electrophoresis anti-
The affine degree of body.Then it takes pictures again to gel after being dyed with Coomassie blue G250 (Biorad).
5 cancer cell Inhibition test of embodiment
Human breast carcinoma BT-474 cell line is being supplemented with 10%FBS (Gibco, USA), 100U/mL penicillin and 100 μ g/
Growth in the RPMI-1640 culture medium (Gibco, USA) of mL streptomysin.5%CO of all cells at 37 DEG C2It is trained in humidification atmosphere
It supports, updates culture medium daily, and use 0.25% trypsase passage cell every three days.A549 is collected by trypsin digestion
Cell, and with 1 × 106The density resuspension of a cell/mL, the 10 μ g shellfishes that addition is separately added into purifying cut down antibody, use film after 84h
Connection albumen V- fluorescein isothiocyanate (FITC) and PI assess the ratio of apoptotic cell for double dyes.
The results show that the inhibition of breast cancer (BT-474) cell pair.Recombination shellfish by the way that purifying is added cuts down antibody culture BT-
474 cells check that cell growth result shows the BT-474 cell well-grown without any processing at the time point of 84h.Phase
Than under, largely (Fig. 4) is disappeared using the cell that the recombination shellfish of purifying cuts down antibody culture.These results indicate that passing through romaine lettuce
The external source shellfish of system Transient Expression cuts down antibody with biological activity and can kill BT-474 cell.Romaine lettuce system is a kind of
Production shellfish cuts down the suitable bioreactor of antibody.
Embodiment 6
Control group: antibody is cut down using zooblast production shellfish；
Experimental group 1: plant shellfish provided by the invention cuts down antibody；
Experimental group 2: antibody is cut down using leaf tobacco production shellfish；
1 shellfish of table cuts down antibody
*Show P≤0.05 compared with the control group；**Show P≤0.01 compared with the control group；
#Show P≤0.05 compared with experimental group 2；##Show P≤0.01 compared with experimental group 2；
As shown in Table 1, experimental group 1 compared with the animal system of control group, cut down by romaine lettuce transient expression shellfish provided by the invention
Antibody, extremely significant (P≤0.01) shorten the production cycle, and extremely significant (P≤0.01) improves protein content, significant (P≤
0.01) protein active is improved, the complexity of protein purification is simplified, extremely significant (P≤0.01) reduces production cost.
For experimental group 1 compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression shellfish cuts down antibody, significant (P≤0.05) contracting
Short production cycle, significant (P≤0.05) improve protein content, and significant (P≤0.05) improves protein active, simplify
The complexity of protein purification, extremely significant (P≤0.01) reduce production cost.
Compared with the control group, tobacco leaf transient expression shellfish cuts down antibody (P≤0.05) more significant than animal system shortening to experimental group 2
Production cycle, significant (P≤0.05) improve protein content, simplify the complexity of protein purification, significant (P≤0.05)
Reduce production cost.In summary test result shows that botanical system especially romaine lettuce system is more economical, efficient table
Up to platform.Can quick transient expression recombinant protein, shellfish can be mass produced in a short time and cut down monoclonal antibody.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Beijing Hui Sheng biotechnology Engineering Co., Ltd
<120>plant cuts down the application in antibody in expression shellfish as host
<130> MP1717044
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1281
<212> DNA
<213>shellfish cuts down heavy chain
<400> 1
atgttcacta actacggaat gaactgggtg agacaggctc caggtaaggg acttgaatgg 60
gttggatgga tcaacactta cactggcgag ccaacttacg ctgccgattt caagaggcgt 120
ttcaccttca gcctcgacac ctctaagtct actgcttacc tccagatgaa ctccctccgt 180
gctgaagata ctgctgtgta ctactgcgct aagtacccac actactacgg ctcatctcac 240
tggtacttcg atgtttgggg acagggaact ctcgtgactg tgtcatctgc ttctactaag 300
ggcccatctg tgttcccact tgctccctca tctaagtcca cttcaggtgg aactgctgct 360
cttggatgcc tcgtgaagga ttactttcca gagccagtga ccgtgtcctg gaattctggt 420
gctcttactt ccggcgttca cactttccca gctgtgcttc aatcttccgg actctactct 480
ctctcctctg ttgtgactgt gccctcttct tcactcggca ctcaaaccta catctgcaac 540
gtgaaccaca agccatccaa caccaaggtg gacaagaagg tcgagccaaa gtcctgcgat 600
aagactcata cttgcccacc atgtccagct ccagaacttc ttggaggacc ttccgtgttt 660
ctgttcccac ctaagcctaa ggacaccctc atgatctcta ggactccaga ggttacatgc 720
gtcgtggttg atgtgtctca tgaggatcct gaggtgaagt tcaactggta cgttgacggt 780
gttgaggtgc acaacgctaa gactaagcca cgtgaggaac agtacaactc cacctacagg 840
gttgtgtctg tgcttactgt gttgcaccag gattggctca acggcaaaga gtacaagtgc 900
aaggtgtcca acaaggctct cccagctcca atcgaaaaga ccatctctaa ggctaagggc 960
cagccaagag agccacaggt ttacactctt ccaccatcca gggaagagat gaccaagaac 1020
caggtgtccc ttacttgcct tgtgaagggc ttctacccat ccgatattgc tgttgagtgg 1080
gagtctaatg gccagcctga gaacaactac aagactactc caccagtgct cgactccgat 1140
ggctcattct tcctgtactc taagctcacc gtggacaagt ctaggtggca gcagggaaat 1200
gtgttctcct gctctgttat gcatgaggcc ttgcacaacc actacaccca gaagtccctt 1260
tcactttccc caggcaagtg a 1281
<210> 2
<211> 455
<212> PRT
<213>shellfish cuts down heavy chain
<220>
<221> MISC_FEATURE
<222> (1)..(455)
<223>X (455)=terminator codon；
<400> 2
Met Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn
20 25 30
Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
35 40 45
Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp
50 55 60
Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
100 105 110
Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
115 120 125
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
130 135 140
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
145 150 155 160
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
165 170 175
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
195 200 205
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
210 215 220
Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
225 230 235 240
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
245 250 255
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
260 265 270
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
275 280 285
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
290 295 300
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
340 345 350
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
355 360 365
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
370 375 380
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
385 390 395 400
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
420 425 430
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
435 440 445
Ser Leu Ser Pro Gly Lys Xaa
450 455
<210> 3
<211> 648
<212> DNA
<213>shellfish cuts down light chain
<400> 3
atggatatcc agatgactca gagccctagc tctctctctg cttctgttgg agatagggtg 60
accatcactt gctccgcttc tcaggacatc tccaactacc tcaactggta tcagcagaag 120
ccaggcaagg ctccaaaggt gttgatctac ttcacctcca gcctccactc tggtgttcca 180
tctaggtttt ccggatctgg ctccggcact gatttcactc tcactatctc tagcctccag 240
ccagaggact tcgctactta ctactgccag cagtactcta ctgtgccatg gactttcgga 300
cagggcacta aggttgagat caagaggact gttgctgccc catccgtgtt cattttccca 360
ccatctgatg agcagctcaa gagcggaact gcttccgttg tttgcctcct caacaacttc 420
tacccacgtg aggctaaggt gcagtggaag gttgacaatg ctctccagtc cggaaactcc 480
caagagtctg ttactgagca ggactccaag gactctacct acagcctcag ctctactctc 540
accctctcta aggctgatta cgagaagcac aaggtgtacg cttgcgaggt tacacaccag 600
ggactttctt caccagtgac caagtctttc aataggggcg agtgctga 648
<210> 4
<211> 216
<212> PRT
<213>shellfish cuts down light chain
<220>
<221> MISC_FEATURE
<222> (1)..(216)
<223>X (216)=terminator codon；
<400> 4
Met Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
1 5 10 15
Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu
35 40 45
Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro
85 90 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys Xaa
210 215
Claims (9)
1. plant cuts down the application in antibody in expression shellfish as host；The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean or small
Wheat；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
2. a kind of expression vector, which is characterized in that the sequence of heavy chain or sequence of light chain and carrier cut down including shellfish.
3. expression vector according to claim 2, which is characterized in that
The sequence of heavy chain or sequence of light chain that the shellfish is cut down be shellfish is cut down to heavy chain, to cut down the codon optimization of light chain be favorite plant to shellfish
Codon, the sequence of light chain that the shellfish of sequence of heavy chain or optimization that the shellfish of the optimization of acquisition is cut down is cut down.
4. expression vector according to claim 3, which is characterized in that the sequence of heavy chain that the shellfish of the optimization is cut down such as SEQ ID
Shown in No.1；The nucleotide sequence for the heavy chain that the shellfish of the optimization is cut down is as shown in SEQ ID No.2；
The sequence of light chain that the shellfish of the optimization is cut down is as shown in SEQ ID No.3；The nucleotides sequence for the light chain that the shellfish of the optimization is cut down
Column are as shown in SEQ ID No.4.
5. according to the described in any item expression vectors of claim 2 to 4, which is characterized in that the carrier is binary plant carrier.
6. expression vector according to any one of claims 2 to 5, which is characterized in that its construction method includes the following steps:
Step 1: shellfish is cut down into heavy chain respectively, shellfish cut down light chain codon optimization be favorite plant codon, obtain:
The sequence of heavy chain that the shellfish of I optimization is cut down；
The sequence of light chain that the shellfish of II optimization is cut down；
Step 2: being separately added into Xbal limit in 5 ' ends of the sequence of light chain that the shellfish of the shellfish of the optimization sequence of heavy chain cut down or optimization is cut down
Property restriction enzyme site processed is separately added into Sac I site in 3 ' ends；
It is cloned into pUC57 carrier, obtains pBeva-H or pBeva-L cloning vector respectively；
Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 respectively by Xbal/Sacl, is cloned into binary plant
Carrier pCam35S obtains expression vector p35S-Beva-H or p35S-Beva-L respectively.
7. cutting down the application in antibody in expression shellfish according to the described in any item expression vectors of claim 2 to 6.
8. a kind of method that plant cuts down antibody as host expresses shellfish, which is characterized in that will be such as any one of claim 2 to 6 institute
The expression vector stated is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen
Matter obtains shellfish and cuts down antibody.
9. according to the method described in claim 8, it is characterized in that, the mediated by agriculture bacillus vacuum infiltration includes the following steps:
Step 1: vacuumizing 25~45s；
Step 2: keeping 30~60s of vacuum (- 95kPa) pressure；
Step 3: releasing stress so that penetrating fluid penetrates into the plant tissue；
It repeats the above steps 2~3 times, is protected from light processing 4d.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710687275.0A CN109385445A (en) | 2017-08-09 | 2017-08-09 | Plant cuts down the application in antibody in expression shellfish as host |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710687275.0A CN109385445A (en) | 2017-08-09 | 2017-08-09 | Plant cuts down the application in antibody in expression shellfish as host |
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| CN109385445A true CN109385445A (en) | 2019-02-26 |
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| CN201710687275.0A Pending CN109385445A (en) | 2017-08-09 | 2017-08-09 | Plant cuts down the application in antibody in expression shellfish as host |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262379A (en) * | 2020-04-29 | 2022-04-01 | 丹生医药技术(上海)有限公司 | PD-1/VEGF tetravalent bispecific antibody, preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031310A (en) * | 2005-04-29 | 2013-04-10 | 开普敦大学 | Expression of proteins in plants |
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2017
- 2017-08-09 CN CN201710687275.0A patent/CN109385445A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031310A (en) * | 2005-04-29 | 2013-04-10 | 开普敦大学 | Expression of proteins in plants |
Non-Patent Citations (3)
| Title |
|---|
| LEI CHEN 等: "Efficient Production of a Bioactive Bevacizumab Monoclonal Antibody Using the 2A Self-cleavage Peptide in Transgenic Rice Callus", 《FRONTIERS IN PLANT SCIENCE》 * |
| VALENTINE NEGROUK等: "Highly efficient transient expression of functional recombinant antibodies in lettuce", 《PLANT SCIENCE》 * |
| 姚文兵: "《生物技术制药概论》", 31 January 2010, 中国医药科技出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114262379A (en) * | 2020-04-29 | 2022-04-01 | 丹生医药技术(上海)有限公司 | PD-1/VEGF tetravalent bispecific antibody, preparation method and application thereof |
| CN114262379B (en) * | 2020-04-29 | 2023-06-02 | 三生国健药业（上海）股份有限公司 | PD-1/VEGF tetravalent bispecific antibody, preparation method and application thereof |
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