CN110092831A - Application of the plant as host in expression A Damu antibody - Google Patents
Application of the plant as host in expression A Damu antibody Download PDFInfo
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- CN110092831A CN110092831A CN201810089048.2A CN201810089048A CN110092831A CN 110092831 A CN110092831 A CN 110092831A CN 201810089048 A CN201810089048 A CN 201810089048A CN 110092831 A CN110092831 A CN 110092831A
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Classifications
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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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- 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
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Abstract
The present invention relates to field of biotechnology, in particular to plant is as host in the application for expressing A Damu antibody.The present invention expresses A Damu monoclonal antibody (Adalimuab, Humira, Xiu Meile) using simple and effective mediated by agriculture bacillus vacuum infiltration methods 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.Recombination A Damu antibody successful expression is determined using SDS-PAGE method.Tumor necrosis factor (tumor necrosis factor, TNF) neutralizes experiments have shown that the A Damu antibody of romaine lettuce production has biological activity.
Description
Technical field
The present invention relates to field of biotechnology, in particular to application of the plant as host in expression A Damu antibody.
Background technique
Rheumatoid arthritis (RA) is a kind of chronic auto-immune disease based on symmetry, multi-joint, Minor articulus,
It is known as the title of " not dead cancer ", the immune system of patient can destroy healthy joint, cause arthralgia, swelling, stiff, generation
Fatigue and powerless symptom, advanced stage can tetanic and lopsided, function be badly damaged, and eventually lead to function of joint and lose.It is tatanic
Rachitis (AS) is that the disease of symptom is wanted based on articulatio sacroiliaca and backbone attachment point inflammation.It is in Qiang Guanlian with HLA-B27.It is certain
Microorganism (such as klebsiella bacillus) and susceptible person autologous tissue have common antigen, can cause abnormal immune response.Tatanic ridge
Column inflammation is four limbs large joint and annulus fibrosus disci intervertebralis and its neighbouring connective fiber and ossification and arthrocleisis is
The chronic inflammatory disease of lesion characteristic.
Rheumatoid arthritis and ankylosing spondylitis are all the higher diseases of disability rate.Adalimumab at present
(Adalimuab, Humira, Xiu Meile) has positive effect to both inflammation.Adalimumab is that full Humanized monoclonal is anti-
One kind of body, and it is a kind of can self-injection biotherapeutics, be by Britain Cambridge Antibody
The first approved tumor necrosis factor α (TNF α) in the whole world of Technology (CAT) and Abbott, U.S. joint research and development is complete
Human monoclonal antibody.The U.S. is used for clinical treatment rheumatoid arthritis and tatanic ridge in approval adalimumab in 2003
Column is scorching.Adalimumab can slow down the progress (x-ray is shown) of patient articular's damage, and can improve physical function, using one
After the section time, the body function and health-related quality of life of patient is significantly improved.
Adalimumab mainly is produced using zooblast at this stage.But animal cell culture needs expensive training
Nutrient solution, stringent workshop condition is complicated for operation, and the time cycle at least two weeks, and also zooblast production capacity is low, production cost
It is high.Sometimes the virus of zooblast institute band can infect the mankind, and safety is low.Therefore it provides a kind of A Damu antibody
Expression has important practical significance.
Summary of the invention
In view of this, the application the present invention provides plant as host in expression A Damu antibody.The present invention utilizes
The efficient platform technology that plant especially romaine lettuce is produced as recombinant protein, expresses A Damu antibody.And in mild item
Active foreign protein is successfully separated out under part, it was demonstrated that plant especially romaine lettuce expression platform can successfully be used to produce Ah reaching
The wooden antibody protein.Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, the potential pest and disease damage of infection human body is eliminated
Deng.Production cost is substantially reduced, Product Safety is improved.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
Application the present invention provides plant as host in expression A Damu antibody.Preferably, the antibody is single
Clonal antibody.The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean, tobacco leaf 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 vectors, sequence of heavy chain or sequence of light chain and carrier including A Damu.
In some specific embodiments of the invention, the sequence of heavy chain or sequence of light chain of the A Damu is by A Damu
Heavy chain, A Damu light chain codon optimization be favorite plant codon, the sequence of heavy chain of the A Damu of the optimization of acquisition or
The sequence of light chain of the A Damu of optimization.
In some specific embodiments of the invention, the sequence of heavy chain of the A Damu of the optimization such as SEQ ID No.1
It is shown；The nucleotide sequence of the heavy chain of the A Damu of the optimization is as shown in SEQ ID No.2；
The sequence of light chain of the A Damu of the optimization is as shown in SEQ ID No.3；The light chain of the A Damu of the optimization
Nucleotide 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: respectively it is the codon of favorite plant by the codon optimization of A Damu heavy chain, A Damu light chain, obtains:
The sequence of heavy chain of the A Damu of I optimization；
The sequence of light chain of the A Damu of II optimization；
Step 2: it is separately added into Xbal restriction enzyme site in 5 ' ends of the sequence of heavy chain of the A Damu of the optimization,
Sac I site is separately added into 3 ' ends；
Xbal restriction enzyme site is added in 5 ' ends of the sequence of light chain of the A Damu of the optimization, adds in 3 ' ends
Enter Sac I site；
It is grand into pUC57 carrier by golden Stryker, pAda-H, pAda-L cloning vector are 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-Ada-H, p35S-Ada-L respectively.Specifically, in order to provide foreign protein
High efficient expression in plant, the present invention is by people A Damu heavy chain and light chain (https: //www.drugbank.ca/
Drugs/DB00051) amino acid sequence obtains core using anti-translation software (https: //www.idtdna.com/CodonOpt)
Nucleotide sequence, and be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.Excellent
5 ' the end of A Damu sequence of heavy chain of change is separately added into Xbal restriction enzyme site, is separately added into the site Sacl in 3 ' ends.
It is separately added into Xbal restriction enzyme site in 5 ' end of A Damu sequence of light chain, is separately added into the site SacI in 3 ' ends.And
It is grand into pUC57 carrier (Fig. 1) by golden Stryker, pAda-H, pAda-L cloning vector are obtained respectively.Genetic fragment passes through
XbaI/Sacl is separated from cloning vector respectively, and is cloned into binary plant carrier pCam35S, is generated plant expression respectively and is carried
Body p35S-Ada-H, p35S-Ada-L (Fig. 2).
The present invention also provides application of the expression vector in expression A Damu antibody.
In addition, a kind of method the present invention also provides plant as host expresses A Damu antibody, the present invention is provided
Total expression vector is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen
Matter obtains A Damu antibody.The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean, tobacco leaf or wheat；The organ of the plant selects
From seed, leaf, rhizome or whole plant.
Specifically, p35S-Ada-L is respectively by using Multiporator by two kinds of plant expression vector p35S-Ada-H
(Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.Equably by obtained strains
It spreads on the selective LB plate of antibiotic containing kanamycin (50mg/L).In the dark after 28 DEG C of incubation 2d, picking list
Colony inoculation is to 0.5L YEB (yeast extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/
LMgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation in oscillator
With 25~28 DEG C of incubation 72h in (220rpm).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) to O.D.600 be 0.5.
It will prepare that mix containing p35S-Ada-H and p35S-Ada-L Agrobacterium equivalent to O.D.600 be 0.5；.It will
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 gently
Ground rotates in bacterial suspension, and drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to take out
Sky, and visual penetration liquid is in leaf tissue.Keep 30~60s of pressure state.It opens the system quickly to release stress, makes
Penetrating fluid penetrates into the space in tissue.The process repeat 2~3 times, until high-visible penetrating fluid spread in romaine lettuce tissue it is bright
It is aobvious.Then romaine lettuce tissue is gently taken out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into plastic foil and covers
In the container of lid.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 pAda-H of the present invention, pAda-L genetic fragment, and construct two kinds of binary plant expression vector p35S-
Ada-H, p35S-Ada-L (Fig. 2), after completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.
After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, and other than firm middle rib region, rest part exists
Vacuum infiltration shows khaki region after 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, is shaken on ice
Dynamic suspension 60min, is centrifuged 15min at 4 DEG C again with 10,000g.Then, liquid is discarded supernatant, sample pellet albumen will be handled
Matter is dissolved in 5mL buffer (20mM KPi, pH 7.8；2mM EDTA；10mM 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, non denatured
The affine degree of antibody is detected in gel electrophoresis.Then gel is clapped again after being dyed with Coomassie blue G250 (Biorad)
According to.
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.Recombinate Ah reaching
The wooden antibody by denaturant gel SDS-PAGE separate we observed in swimming lane estimation molecular weight be respectively about 24kDa and
48kDa (Fig. 3 A), meets the albumen size of A Damu antibody light and weight chain.It is observed about in non denatured gel electrophoresis
The band of 150kDa (Fig. 3 B), it was demonstrated that romaine lettuce recombination light and weight chain is successfully combined into antibody structure, meets A Damu antibody protein
Molecular weight.Based on Bradford measuring method and spectrodensitometry control group measurement purification of samples protein content be about
0.94mg/g。
Tumor necrosis factor α (TNF α) is a kind of naturally occurring cell factor, is related to normal inflammation and immune response.Class
It is found in the synovial membrane of rheumatoid arthritis, including juvenile idiopathic arthritis, psoriatic arthritis and patients with ankylosing spondylitis
TNF level increases.It has also been found that TNF level increases in psoriasis (Ps) patch.Adalimumab be specifically bound to TNF-α and
It and p55 and p75 cell surface receptor is blocked to interact.Elisa assay adalimumab Fab segment can be in conjunction with TNF α.
TNF α is coated with plate as substrate, and the anti-light chain IgG of goat-anti people IgK specificity with HRP label can specificity and Fab segment knot
It closes, display Fab segment and TNF α have preferably in conjunction with (Fig. 4).These results indicate that passing through the external source of romaine lettuce system Transient Expression
A Damu antibody has biological activity and can neutralize TNF α.The result shows that plant especially romaine lettuce is a kind of production Ah reaching
The suitable bioreactor of the wooden antibody.
The present invention is using romaine lettuce come transient expression A Damu antibody, and (4d) can produce the egg of high-content in a relatively short period of time
White matter.Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And it is this
Method reduces bio-safety problem to the maximum extent because processed romaine lettuce tissue be usually in completely enclosed facility or
It is developed in container, biological pollution problem is not present.Romaine lettuce is substantially free of plant noxious material, and itself fiber is few, benefit
Protein purification in downstream.Using romaine lettuce system production A Damu monoclonal antibody, production cycle and production can be greatly shortened
Cost.
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 Ah Da wood plant binary expression vector p35S-Ada-H (heavy chain) and p35S-Ada-L (light chain) building stream
Journey；Using restriction enzyme (Xbal/SacI) double digestion, cut Ah 's wood H heavy chain respectively from Fig. 1 cloning vector, connect into
The site Xbal/SacI of pCam35S generates plant binary expression vector p35S-Ada-H；Utilize restriction enzyme (Xbal/
SacI) double digestion cuts A Damu antibody light chain from Fig. 1 cloning vector respectively, and heavy chain connects the Xbal/SacI into pCam35S
Site generates plant binary expression vector p35S-Ada-L, p35S-Ada-H；
LB and RB:Ti plasmid right boundary；35S, the CaMV 35S with tobacco mosaic virus (TMV) (TMV) 5 ' UTR start
Son；NPT II, the expression of the coding nptII gene for kalamycin resistance；Nos3 ', terminator；
Fig. 3 (A) shows PAGE gel electrophoresis result；Swimming lane 1: the A Damu recombinant antibodies of romaine lettuce expression；Swimming lane 2: quotient
Industry A Damu recombinant antibodies；Fig. 3 (B) shows native gel electrophoresis result；Swimming lane 3: the A Damu recombinant antibodies of romaine lettuce expression；Swimming
Road 4: business A Damu recombinant antibodies；
Fig. 4 shows through ELISA test experience research shows that tumor necrosis factor α (TNF can be neutralized by purifying A Damu antibody
α), there is significant biological activity.
Specific embodiment
Application the invention discloses plant as host in expression A Damu antibody, those skilled in the art can borrow
Reflect 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
It is it will be apparent that they are considered as being included in the present invention for technical staff.Method and application of the invention has passed through
Preferred embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to described herein
Methods and applications be 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, which can use, is mass produced A Damu monoclonal antibody in the romaine lettuce system short time.
Plant provided by the invention raw materials used and reagent in the application in expression A Damu antibody as host
It is bought by market.
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's A Damu heavy chain of antibody, light chain, (https: //
Www.drugbank.ca/drugs/DB00051) amino acid sequence using anti-translation software (https: //
Www.idtdna.com/CodonOpt nucleotide sequence) is obtained, and is the codon of favorite plant by its codon optimization, by
Jin Sirui company (Nanjing, China) synthesis.The restricted digestion of Xbal is separately added into the end A Damu sequence of heavy chain 5' of optimization
Site is separately added into the site SacI in the end 3'.The restricted digestion position Xbal is separately added into the end A Damu sequence of light chain 5'
Point is separately added into the site Sacl in the end 3'.And (Fig. 1) is cloned into pUC57 carrier by Jin Sirui company, it generates respectively
PAda-H, pAda-L cloning vector.Genetic fragment is separated from cloning vector respectively by Xbal/Sacl, and is cloned into double base
Plant vector, pCam35S generate plant expression vector p35S-Ada-H, p35S-Ada-L respectively.By two kinds of plant expression vectors
Respectively by with Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation to Agrobacterium tumefaciens
In GV3101.Obtained strains are equably spread on the selective LB plate of antibiotic containing kanamycin (50mg/L).?
In dark after 28 DEG C of incubation 2d, picking single colonie is inoculated into 0.5L YEB (yeast extract meat soup, 5g/L sucrose, 5g/L pancreas egg
White peptone, 6g/L yeast extract, 0.24g/L MgSO4, pH7.2) and supplement antibiotic liquid culture medium (that is mould for 50mg/L card
Element).By the culture of inoculation with 25~28 DEG C of incubation 72h in oscillator (220rpm).Pass through addition YEB culture medium measurement
OD600 value is simultaneously 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 pAda-H, pAda-L genetic fragment (Fig. 2), and constructs two kinds of binary plant expression vector p35S-
Ada-H, p35S-Ada-L.After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.Infiltration
Afterwards, most romaine lettuce groups are flooded during being woven in vacuum immersion, and other than firm middle rib region, rest part is in vacuum
Infiltration showed khaki region after 4 days.
The vacuum infiltration of 2 mediated by agriculture bacillus of embodiment
It will prepare that mix containing p35S-Ada-H and p35S-Ada-L Agrobacterium equivalent to O.D.600 be 0.5.It will
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 gently
Ground rotates in bacterial suspension, and drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to take out
Sky, and visual penetration liquid is in leaf tissue.It is kept for pressure state 30~60 seconds.Open the system quickly to release stress,
Penetrating fluid is set to penetrate into the space in tissue.The process repeats 2 to 3 times, until high-visible penetrating fluid is spread in romaine lettuce tissue
Obviously.Then romaine lettuce tissue is gently taken out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into plastic foil
In the container of 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.Collect supernatant
Liquid is mixed with ammonium sulfate (50%), and shakes be incubated for 60 minutes on ice.Divided again at 4 DEG C by centrifuge (10,000g)
From 15 minutes.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, shakes suspend 60 minutes on ice, again 4
With 10,000g centrifugation 15 minutes at DEG C.Then, liquid is discarded supernatant, processing sample pellet protein is dissolved in 5mL buffer
(20mM KPi, pH 7.8；2mM EDTA；10mM 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.
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).It is anti-to recombinate Ah 's wood
Body separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about 24kDa and 48kDa item in swimming lane
Band (Fig. 3 A), meets that A Damu antibody is light, the albumen size of heavy chain.About 150kDa is observed in non denatured gel electrophoresis
The band of (Fig. 3 B), it was demonstrated that romaine lettuce recombination light and weight chain is successfully combined into antibody structure, meets A Damu antibody protein molecular weight.
Protein content based on Bradford measuring method and spectrodensitometry control group measurement purification of samples is about 0.94mg/g.
5 cancer cell Inhibition test of embodiment
Tumor necrosis factor α (TNF α) is a kind of naturally occurring cell factor, is related to normal inflammation and immune response.Class
It is found in the synovial membrane of rheumatoid arthritis, including juvenile idiopathic arthritis, psoriatic arthritis and patients with ankylosing spondylitis
TNF level increases.It has also been found that TNF level increases in psoriasis (Ps) patch.Adalimumab be specifically bound to TNF-α and
It and p55 and p75 cell surface receptor is blocked to interact.Elisa assay adalimumab Fab segment can be in conjunction with TNF α.
TNF α is coated with plate as substrate, and the anti-light chain IgG of goat-anti people IgK specificity with HRP label can specificity and Fab segment knot
It closes, display Fab segment and TNF α have preferably in conjunction with (Fig. 4).These results indicate that passing through the external source of romaine lettuce system Transient Expression
A Damu antibody has biological activity and can neutralize TNF α.In conjunction with dynamics pass through surface plasma body resonant vibration measure
It proves, the adalimumab in romaine lettuce source is in conjunction with the ka of the soluble TNF α of three different batches of business adalimumab
Rate constant, k d dissociation rate constant, Kd dissociation equilibrium binding constant are substantially similar (table 1).The result shows that plant
Especially romaine lettuce is a kind of suitable bioreactor for producing A Damu antibody.
1 romaine lettuce of table expresses the combination of adalimumab (experimental group) and business adalimumab (control group) and sTNF α
Kinetic results
The dynamics that the above results display combines is measured by surface plasma body resonant vibration, tests the three of each test monoclonal antibody
A uniqueness batch.
Note: ka association rate constant, kd dissociation rate constant, Kd dissociation equilibrium binding constant, ms milliseconds, s seconds, sTNF α
Soluble tumor necrosis factor α.
Embodiment 6
Experimental group: plant production A Damu antibody provided by the invention；
Control group 1: A Damu antibody is produced using zooblast；
Experimental group 2: leaf tobacco production A Damu antibody is utilized；
2 A Damu antibody of table
*Show P≤0.05 compared with control group 1；**Show P≤0.01 compared with control group 1；
#Show P≤0.05 compared with experimental group 2；##Show P≤0.01 compared with experimental group 2；
As shown in Table 2, experimental group 1 is compared with the animal system of control group 1, romaine lettuce transient expression Ah reaching provided by the invention
The wooden antibody, extremely significant (P≤0.01) shorten the production cycle, and extremely significant (P≤0.01) improves protein content, extremely significant (P≤
0.01) reduce with TNF α binding constant (Kd), simplify the complexity of protein purification, extremely significant (P≤0.01) reduces
Production cost.
Experimental group 1 is compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression A Damu antibody, significant (P≤0.05) contracting
The short production cycle, significant (P≤0.05) reduce with TNF α binding constant (Kd), significant (P≤0.05) reduces and TNF α
Binding constant (Kd), simplifies the complexity of protein purification, and extremely significant (P≤0.01) reduces production cost.
Compared with the control group, tobacco leaf transient expression A Damu antibody (P≤0.05) more significant than animal system shortens experimental group 2
Production cycle, significant (P≤0.05) reduce with TNF α binding constant (Kd), simplify the complexity of protein purification, show
Writing (P≤0.05) reduces production cost.
In summary test result show botanical system especially romaine lettuce system be it is more economical, efficiently express platform.
Can quick transient expression recombinant protein, A Damu Antibodies Monoclonal antibodies can be mass produced in a short time.
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 table
<110>converge in the farsighted sincere sea in Beijing healthy Science and Technology Ltd.
<120>application of the plant as host in expression A Damu antibody
<130> MP1724894
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1359
<212> DNA
<213>A Damu heavy chain (heavy chain of Ada)
<400> 1
atggaagtgc agctcgtaga atccggtggc gggctggttc aaccggggag atcattacgt 60
ttatcatgtg ctgcttctgg gttcacattt gatgattacg ccatgcactg ggtgagacag 120
gccccaggca agggacttga atgggtaagt gccattacct ggaatagtgg gcatatcgac 180
tatgctgatt ctgtcgaggg acgtttcact attagcagag acaacgctaa aaactccctg 240
tacctgcaga tgaattctct ccgagcagag gacacagccg tctactactg tgcaaaagta 300
agctacctga gcacagccag ctcccttgat tactggggcc aagggacttt ggtgaccgtg 360
agcagtgcct ctactaaagg tccctcagta ttcccgctgg ccccttccag caagagtacc 420
tcaggtggca cagccgccct gggatgctta gtgaaagact attttccaga accggtcacc 480
gtatcatgga attcaggggc tctcacatct ggtgtgcata cttttccggc cgtgcttcaa 540
tcctccgggc tctactctct tagtagcgta gtcactgtgc cctcctcttc cttaggtact 600
cagacttata tctgtaatgt caaccataag cccagtaaca caaaagttga taaaaaagtt 660
gagcccaaaa gttgtgataa aacacatact tgtccacctt gtcccgcacc cgaattgtta 720
ggaggtccta gtgttttttt gtttcccccc aaaccgaaag atactcttat gatctcaagg 780
actccagaag ttacttgtgt ggtcgtcgat gtctctcatg aagaccccga agtgaagttt 840
aacgtatacg tcgacggtgt cgaagttcac aatgccaaaa ctaagcctcg tgaagaacaa 900
tacaacagta catatagggt cgtgagtgtg ctcacggtgc tgcaccaaga ctggttaaac 960
ggcaaggagt acaagtgcaa ggtgtctaac aaggctctgc ctgcacctat tgaaaaaact 1020
atctcaaaag caaaaggaca gcctagggag cctcaggtct atacactccc tccatctagg 1080
gatgaattga cgaagaatca ggtttcactc acttgccttg tcaagggatt ctatccttcc 1140
gatattgccg tggaatggga gagtaacggg cagccggaaa ataactataa aacgacaccc 1200
ccagtgctcg actccgacgg gagctttttt ctttatagta agcttactgt ggataagtct 1260
cgttggcaac aaggcaatgt gttctcctgt agtgtcatgc acgaagcctt gcataaccac 1320
tatacccaaa aaagcctcag tctctcaccc ggcaaataa 1359
<210> 2
<211> 451
<212> PRT
<213>A Damu heavy chain (heavy chain of Ada)
<400> 2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60
Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Val Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 3
<211> 645
<212> DNA
<213>A Damu light chain (light chain of Ada)
<400> 3
atggacattc agatgacgca atctccaagc agcctgtccg catctgtggg tgaccgtgtc 60
acgatcacct gccgagccag tcagggaata cgaaactacc tcgcctggta tcaacagaag 120
cccggaaaag ccccaaaatt gttgatctac gccgccagca ctctgcaatc tggagtaccc 180
agtcgtttct ccgggagcgg gagcggcaca gacttcactc tcacgatctc ctcccttcaa 240
ccagaagatg tagccactta ttactgccaa aggtacaatc gagcacctta cacattcggc 300
cagggaacca aggtagaaat aaagagaaca gtcgcagctc cctctgtgtt tatattcccg 360
ccgtctgatg aacagctgaa atctggtacg gcctcagtag tctgcctttt aaataacttc 420
tatccgcgag aagccaaggt acagtggaag gtagacaatg ccctccagtc tggcaattcc 480
caggagtctg ttaccgaaca ggactctaaa gactctacgt attccctgtc ttccacactg 540
actctttcaa aagctgatta cgagaagcac aaggtatatg cttgcgaggt aacgcaccaa 600
ggcttgtcaa gcccagtaac gaagtcattc aaccgtggtg aatgc 645
<210> 4
<211> 214
<212> PRT
<213>A Damu light chain (light chain of Ada)
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg Ala Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
Claims (9)
1. application of the plant as host in expression A Damu antibody；The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean, cigarette
Leaf or 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 sequence of heavy chain or sequence of light chain and carrier including A Damu.
3. expression vector according to claim 2, which is characterized in that the sequence of heavy chain or sequence of light chain of the A Damu be
It is the codon of favorite plant, the weight of the A Damu of the optimization of acquisition by the codon optimization of A Damu heavy chain, A Damu light chain
The sequence of light chain of chain-ordering or the A Damu of optimization.
4. expression vector according to claim 3, which is characterized in that the sequence of heavy chain of the A Damu of the optimization such as SEQ
Shown in ID No.1；The nucleotide sequence of the heavy chain of the A Damu of the optimization is as shown in SEQ ID No.2；
The sequence of light chain of the A Damu of the optimization is as shown in SEQ ID No.3；The nucleosides of the light chain of the A Damu of the optimization
Acid sequence is 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: respectively it is the codon of favorite plant by the codon optimization of A Damu heavy chain, A Damu light chain, obtains:
The sequence of heavy chain of the A Damu of I optimization；
The sequence of light chain of the A Damu of II optimization；
Step 2: Xbal restriction enzyme site is separately added into 5 ' ends of the sequence of heavy chain of the A Damu of the optimization, 3 '
End is separately added into Sac I site；
Xbal restriction enzyme site is added in 5 ' ends of the sequence of light chain of the A Damu of the optimization, is added in 3 ' ends
Sac I site；
It is cloned into pUC57 carrier, obtains pAda-H, pAda-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-Ada-H, p35S-Ada-L respectively.
7. according to application of the described in any item expression vectors of claim 2 to 6 in expression A Damu antibody.
8. a kind of method of plant as host expresses A Damu antibody, which is characterized in that will be such as any one of claim 2 to 6
The expression vector is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen
Matter obtains A Damu antibody；
The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean, tobacco leaf or wheat；The organ of the plant is selected from seed, leaf, rhizome
Or whole plant.
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.
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| PCT/CN2018/116153 WO2019148939A1 (en) | 2018-01-30 | 2018-11-19 | Use of plant as host in expression of adalimumab antibody |
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| CN107635581A (en) * | 2015-02-13 | 2018-01-26 | 赛诺菲 | Stable liquid preparation for monoclonal antibody |
| CN107354171A (en) * | 2016-05-10 | 2017-11-17 | 美迪西普亚医药科技(上海)有限公司 | Recombinate preparation method of the adalimumab Fab fragments in insect cell expression system |
| CN107058377A (en) * | 2017-06-16 | 2017-08-18 | 深圳惠升生物科技有限公司 | Application of the plant as host in the vaccine of expression Middle East respiration syndrome |
| CN107083398A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in the expression antibody of PD 1 and/or PD L1 antibody |
| CN107236759A (en) * | 2017-06-16 | 2017-10-10 | 深圳惠升生物科技有限公司 | Application of the romaine lettuce as host in expressing protein and/or polypeptide |
| CN107254486A (en) * | 2017-06-16 | 2017-10-17 | 深圳惠升生物科技有限公司 | Application of the romaine lettuce as host in expression growth factor |
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| WO2019148939A1 (en) | 2019-08-08 |
| CN110092831B (en) | 2022-05-20 |
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