CN110511276A - Application of the plant as host in expression Aducanumab antibody - Google Patents
Application of the plant as host in expression Aducanumab antibody Download PDFInfo
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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 Aducanumab antibody.The present invention expresses Aducanumab antibody 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 is infected 4 days.Aducanumab antibody successful expression is determined using Western Blot protein hybridization method, goes out Aducanumab monoclonal antibody with AKTA protein purification system successful purification.Active testing is the result shows that Aducanumab significantly reduces the accumulation of amyloid protein (A β) in Tg2576 transgenic mice brain.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant is as host in expression Aducanumab antibody
Using.
Background technique
Alzheimer's disease (AD) is the most common cause of disease of dementia, and in the U.S., there are about 4,500,000 people to infect, the whole world there are about
26600000 people.The pathological characters of this disease are the intracellular nerves of extracellular accumulation β-amyloid (Α β) patch and brain
Fibrinogen tangles.Diagnosis is to exclude other dull-witted originals by the clinical assessment of nuerological and neuropsychic symptom to AD
Cause.By simply recognizing screening test (MMSE), AD is typically divided into slight, moderate and severe stage.Approved inhibition
Acetylcholinesterase (AChE) activity or the drug therapy of antagonism intracerebral n- methyl-d-aspartate receptor may temporarily improve certain
The AD symptom of a little patients, but not change the progress of disease.
The inherent cause of early stage and Delayed onset familial AD have obtained sufficient document and have recorded.ApoE4 allele with
Delayed onset familial and sporadic AD are closely related, it was reported that the gene frequency of AD patient is 50%-65%, about generally
Three times of crowd and other patient with nervous system disease gene frequencies.In addition to AD, ApoE4 allele also with other starch
Sample albumen formation obstacle is related, including Cerebral amyloid angiopathy (CAA).Therefore, the patient for carrying ApoE4 allele can
The crowd that the cause of disease is different in AD patient can be represented.
Extracellular amyloid plaques are one of AD significant pathology discoveries in deposited in the brain, 1906
AloisAlzheimer is reported for the first time.These amyloid plaques mainly form the sequence generated cream by amyloid protein (A β) polypeptide
The amyloid precusor protein of ditch passes through the activity of β and gamma-secretase.Amyloid protein (A β), especially its oli-gomeric forms,
It is toxic to neuron, it is considered to be the inducement of AD.The therapy for reducing amyloid protein (A β) level in brain may subtract
Light cognition dysfunction, and prevent further synaptic loss, axonal degeneration and Neuronal cell death.Amyloid protein (A β)
It can energetically be transported by blood-brain barrier, in the mouse model of AD, systematic beta amyloid protein antibody increases blood plasma
Beta amyloid protein content, while reducing level and passing through different mechanism in central nervous system (CNS), including dismissing brain
Beta amyloid protein patch swallows the beta amyloid protein of conditioning, finally by the beta amyloid protein of outflow from brain
Beta amyloid protein caused by balance change.
Exist for the antibody A ducanumab of beta amyloid fibrinogen (anti-amyloid beta protofibril)
In one II clinical trial phase BAN2401-G000-201,18 months results of study of final early stages alzheimer's disease patient, which reach, is ground
Study carefully terminal, has significantly delayed the progress of stages alzheimer's disease to score, decreased the accumulation of amyloid protein in brain.
Plant transient expression technology is will to contain mesh using a variety of different technical approach in plant growth to certain phase
The plasmid of mark albumen is transferred in plant cell, and efficient, controllable expression system is established in plant cell, it is short to obtain the gene
The technology of temporary controlled expression.It is short the time required to transient expression compared with stablizing expression, it does not need exogenous origin gene integrator to place
In main plant chromosome, it is only necessary to which several days time can take experimental result.Compared with bacterial expression system, plant expression system
Expressed albumen can correctly be folded, process, modify, protein active produced is higher than bacterial expression system；
Compared with animal cell expression system, the cost of plant expression system is very low, only its one thousandth to 2/1000ths.
Summary of the invention
In view of this, the application the present invention provides plant as host in expression Aducanumab antibody.The present invention
The efficient platform technology produced using plant especially romaine lettuce as recombinant protein, expresses Aducanumab antibody.And
Active foreign protein is successfully separated out under conditions of mild, it was demonstrated that plant especially romaine lettuce expression platform can successfully be used to
Produce Aducanumab antibody protein.Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, infection human body is eliminated
Potential pest and disease damage etc..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 Aducanumab antibody.Preferably, the antibody
For monoclonal antibody.The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The plant
The organ of object is 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 including Aducanumab antibody and
Carrier.
In some specific embodiments of the invention, the sequence of heavy chain or sequence of light chain of the Aducanumab antibody are
It is the codon of favorite plant by the codon optimization of Aducanumab heavy chain of antibody, Aducanumab antibody light chain, acquisition
The sequence of light chain of the Aducanumab antibody of the sequence of heavy chain or optimization of the Aducanumab antibody of optimization.
In some specific embodiments of the invention, the amino acid sequence of the heavy chain of the Aducanumab antibody of the optimization
Column are as shown in SEQ ID No.2；The nucleotide sequence of the heavy chain of the Aducanumab antibody of the optimization such as SEQ ID No.1 institute
Show；
The amino acid sequence of the light chain of the Aducanumab antibody of the optimization is as shown in SEQ ID No.4；The optimization
Aducanumab light chain nucleotide sequence as shown in SEQ ID No.3.
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: being respectively that plant is inclined by the codon optimization of Aducanumab heavy chain of antibody, Aducanumab antibody light chain
Good codon obtains:
The sequence of heavy chain of the Aducanumab antibody of I optimization；
The sequence of light chain of the Aducanumab antibody of II optimization；
Step 2: it is restricted to be separately added into Xbal in 5 ' ends of the sequence of heavy chain of the Aducanumab antibody of the optimization
Restriction enzyme site is separately added into Sac I site in 3 ' ends；
Xbal restriction enzyme site, In is added in 5 ' ends of the sequence of light chain of the Aducanumab antibody of the optimization
Sac I site is added in 3 ' ends；
It is grand into pUC57 carrier by golden Stryker, pAduca-H, pAduca-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-Aduca-H, p35S- Aduca-L respectively.
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by people's Aducanumab antibody weight
Chain and light chain (https: //www.genome.jp/dbget-bin/www_bget dr:D10541) amino acid sequence utilizes
Anti- translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) obtains nucleotide sequence,
And be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.In optimization
5 ' end of Aducanumab antibody heavy chain sequence is separately added into Xbal restriction enzyme site, is separately added into Sacl in 3 ' ends
Point.It is separately added into Xbal restriction enzyme site in 5 ' end of Aducanumab antibody light chain sequences, is separately added into 3 ' ends
The site SacI.And it is grand into pUC57 carrier by golden Stryker, pAduca-H, pAduca-L cloning vector (Fig. 1) are obtained respectively.
Genetic fragment is separated from cloning vector respectively by XbaI/Sacl, and is cloned into binary plant carrier pCam35S, is produced respectively
Plant expression vector p35S-Aduca-H, p35S-Aduca-L (Fig. 2).
The present invention also provides application of the expression vector in expression Aducanumab antibody.
In addition, a kind of method the present invention also provides plant as host expresses Aducanumab antibody, it will be of the invention
The expression vector of offer is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen
Matter obtains Aducanumab antibody.
Specifically, two kinds of plant expression vectors p35S-Aduca-H, p35S-Aduca-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).It incubates for 28 DEG C in the dark
After educating 2d, picking single colonie is inoculated into 0.5LYEB (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 training of inoculation
Object is supported 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.
It is 0.5 that the p35S-Aduca-H that contains that will be prepared, p35S-Aduca-L Agrobacterium equivalent, which are mixed to O.D.600,；
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 light
It lightly rotates in bacterial suspension, drier is sealed.By vacuum pump (Welch Vacuum, Niles, IL, USA) open with
It evacuates, and visual penetration liquid is in leaf tissue.Keep 30~60s of pressure state.Open the system quickly to release stress,
Penetrating fluid is set to penetrate into the space in tissue.The process repeats 2~3 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 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 pAduca-H of the present invention, pAduca-L genetic fragment (Fig. 1), and construct two kinds of binary plant expression
Carrier p35S-Aduca-H, p35S-Aduca-L (Fig. 2), after completing construct, being digested with specific restriction enzyme confirms gene
Segment is complete.After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, in addition to firm middle rib region
Outside, rest part 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；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, 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
Aducanumab antibody separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is respectively about in swimming lane
23kDa and 50kDa (Fig. 3 left figure, swimming lane 2), with business Aducanumab antibody treadbelt (Fig. 3 left figure, swimming lane in the same size
3), meet the albumen size of Aducanumab antibody light and weight chain.Rather than without discovery band in the romaine lettuce extracting solution infected.
The band of about 150kDa (Fig. 3 right figure, swimming lane 2) is observed in non denatured gel electrophoresis, it was demonstrated that romaine lettuce recombinates light and weight chain
It successfully is combined into antibody structure, meets Aducanumab antibody protein molecular weight (Fig. 3 right figure, swimming lane 3, business
Aducanumab antibody).Protein content based on Bradford measuring method and spectrodensitometry control group measurement purification of samples is big
About 1.85mg/g.
Using the activity of Tg2576 transgenic mice evaluation Aducanumab antibody.In the transgenic mice for carrying patch
After chronic administration, the exposure in blood plasma and brain is related to dose linear.Soluble and monomeric and widow are extracted using diethylamine (DEA)
The A β 40 and A β 42 of dimer form, testing result show dose-dependently to reduce the A β measured in brain homogenate relative to carrier pair
According to reduction up to 50%.
The present invention is using romaine lettuce come transient expression Aducanumab antibody, and (4d) can produce height and contain in a relatively short period of time
The protein of amount.Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And
And this method reduces bio-safety problem to the maximum extent, because processed romaine lettuce tissue is usually completely enclosed
It is developed in facility or container, biological pollution problem is not present.Romaine lettuce does not contain plant noxious material, and itself protein content
It is few, conducive to the protein purification in downstream.Using romaine lettuce system production Aducanumab monoclonal antibody, production week can be greatly shortened
Phase and production 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 pAduca-H and pAduca-L schematic diagram；
Fig. 2 shows that Aducanumab plant binary expression vector p35S-Aduca-H (heavy chain) and p35S- Aduca-L are (light
Chain) building process；Using restriction enzyme (Xbal/SacI) double digestion, cut respectively from Fig. 1 cloning vector
Aducanumab heavy chain connects the site Xbal/SacI into pCam35S, generates plant binary expression vector p35S-Aduca-H；
Using restriction enzyme (Xbal/SacI) double digestion, Aducanumab antibody light chain is cut respectively from Fig. 1 cloning vector, weight
Chain connects the site Xbal/SacI into pCam35S, generates plant binary expression vector p35S-Aduca-L；
LB andRB: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；Nos 3 ', terminator；
Fig. 3 shows electrophoresis result；Wherein Fig. 3 left figure shows PAGE gel electrophoresis result；Fig. 3 right figure shows native gel electricity
Swimming result；Swimming lane 1: non-to infect romaine lettuce；Swimming lane 2: the Aducanumab antibody of romaine lettuce expression；Swimming lane 3:Aducanumab antibody
Positive control；
Fig. 4 shows through neutralization experiments have shown that inhibition of the Aducanumab of romaine lettuce purifying to beta-amyloid protein, has aobvious
The biological activity of work.
Specific embodiment
Application the invention discloses plant as host in expression Aducanumab antibody, those skilled in the art can
To use for reference present disclosure, it is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this
It is it will be apparent that they are considered as being included in the present invention for the technical staff of field.Method and application of the invention is
Be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to herein
The 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, which can use, is mass produced Aducanumab monoclonal antibody in the romaine lettuce system short time.
Plant provided by the invention raw materials used and reagent in the application in expression Aducanumab antibody as host
It is available on the market.
Below with reference to embodiment, the present invention is further explained:
The building of 1 plant transient expression vector of embodiment
For the high efficient expression by foreign protein in plant, by Aducanumab heavy chain of antibody, light chain, (https: //
Www.genome.jp/dbget-bin/www_bget dr:D10541) amino acid sequence using anti-translation software (https: //
Www.ebi.ac.uk/Tools/st/emboss_backtranseq/ nucleotide sequence) is obtained, and is by its codon optimization
The codon of favorite plant, by Jin Sirui company (Nanjing, China) synthesis.At the end Aducanumab sequence of heavy chain 5' of optimization
End is separately added into Xbal restriction enzyme site, is separately added into the site SacI in the end 3'.In Aducanumab sequence of light chain 5'
End is separately added into Xbal restriction enzyme site, is separately added into the site Sacl in the end 3'.And it is cloned by Jin Sirui company
In pUC57 carrier, pAduca-H, pAduca-L cloning vector (Fig. 1) are obtained respectively, and genetic fragment is distinguished by Xbal/Sacl
It is separated from cloning vector, and is cloned into binary plant carrier, pCam35S generates plant expression vector p35S-Aduca- respectively
H, p35S-Aduca-L (Fig. 2).By two kinds of plant expression vectors pass through respectively with Multiporator (Eppendorf,
Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over containing card
On the selective LB plate of that mycin antibiotic (50mg/L).In the 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 tryptone, 6g/L yeast extract, 0.24g/LMgSO4,
PH7.2) and antibiotic liquid culture medium (50mg/L kanamycins) is supplemented.By the culture of inoculation at oscillator (220rpm)
In 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 is collected
Liquid is centrifuged (4500 revolving speed) 10min.Agrobatcerium cell is resuspended in osmotic medium (10mM MES, 10 mM MgSO4) in extremely
O.D.600 is 0.5.
The vacuum infiltration of 2 mediated by agriculture bacillus of embodiment
It will prepare to mix containing p35S-Aduca-H and p35S-Aduca-L Agrobacterium equivalent to O.D.600 and be
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；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).Recombination
Aducanumab antibody separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about 23kDa in swimming lane
And 50kDa band (Fig. 3 left figure), meet that Aducanumab antibody is light, the albumen size of heavy chain.In non denatured gel electricity
The band of about 150kDa (Fig. 3 right figure) is observed in swimming, it was demonstrated that romaine lettuce recombination light and weight chain is successfully combined into antibody structure, accords with
Close Aducanumab antibody protein molecular weight.Purification of samples is measured based on Bradford measuring method and spectrodensitometry control group
Protein content is about 1.85mg/g.
5 Aducanumab of embodiment is to beta-amyloid protein Activity determination
Using Tg2576 transgenic mice, age of mouse is 9.5~15.5 months.It is small with Aducanumab injection injection transgenosis
Mouse, injection dosage are respectively 0.3mg/kg, 1mg/kg, 3mg/kg, 10mg/kg, 30mg/kg；Injection is primary weekly, continuous to inject
10 weeks, 24 mouse of co-injection.Test mice brain beta-amyloid protein is extracted after injection, and quantitative with ELISA method.Test
Data carry out variance analysis with SPSS 22.0.Carried in the transgenic mice of patch after chronic administration as the result is shown, blood plasma and
Exposure in brain is related to dose linear.The A β 40 and A β of soluble and monomeric and oligomeric forms are extracted using diethylamine (DEA)
42, testing result shows that dose-dependently reducing the A β measured in brain homogenate reduces up to 50% relative to vehicle Control.
The present invention is using romaine lettuce come transient expression antibody, and (4d) can produce the protein of high-content in a relatively short period of time.
Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this method is most
Reduce to limits bio-safety problem, because processed romaine lettuce tissue is usually in completely enclosed facility or container
Biological pollution problem is not present in exploitation.Romaine lettuce does not contain plant noxious material, and itself protein content is few, is conducive to downstream
Protein purification.Using romaine lettuce system production monoclonal antibody, production cycle and production cost can be greatly shortened.
Embodiment 6
Control group: Aducanumab antibody is produced using zooblast；
Experimental group 1: plant production Aducanumab antibody provided by the invention；
Experimental group 2: leaf tobacco production Aducanumab antibody is utilized；
1 Aducanumab antibody of table
*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 is compared with the animal system of control group, romaine lettuce transient expression provided by the invention
Aducanumab antibody, extremely significant (P≤0.01) shorten the production cycle, and extremely significant (P≤0.01) improves protein content,
Significantly (P≤0.01) improves protein active, simplifies the complexity of protein purification, and extremely significant (P≤0.01) reduces life
Produce cost.
Experimental group 1 is compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression Aducanumab antibody, significant (P≤
0.05) production cycle is shortened, significant (P≤0.05) improves protein content, and significant (P≤0.05) improves protein active,
The complexity of protein purification is simplified, extremely significant (P≤0.01) reduces production cost.
Compared with the control group, tobacco leaf transient expression Aducanumab antibody is more significant than animal system (P≤0.05) for experimental group 2
The production cycle is shortened, the complexity of protein purification is simplified, significant (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.It being capable of quick transient expression
Aducanumab monoclonal antibody can be mass produced in recombinant protein 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>Wang Yueju
<120>application of the plant as host in expression Aducanumab antibody
<130> MP1916308
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1359
<212> DNA
<213>heavy chain (heavy chain of Aducanumab) of Aducanumab
<400> 1
atggtgcagc tggtggagag cggcggcggc gtggtgcagc ccggcaggag cctgaggctg 60
agctgcgccg ccagcggctt cgccttcagc agctacggca tgcactgggt gaggcaggcc 120
cccggcaagg gcctggagtg ggtggccgtg atctggttcg acggcaccaa gaagtactac 180
accgacagcg tgaagggcag gttcaccatc agcagggaca acagcaagaa caccctgtac 240
ctgcagatga acaccctgag ggccgaggac accgccgtgt actactgcgc cagggacagg 300
ggcatcggcg ccaggagggg cccctactac atggacgtgt ggggcaaggg caccaccgtg 360
accgtgagca gcgccagcac caagggcccc agcgtgttcc ccctggcccc cagcagcaag 420
agcaccagcg gcggcaccgc cgccctgggc tgcctggtga aggactactt ccccgagccc 480
gtgaccgtga gctggaacag cggcgccctg accagcggcg tgcacacctt ccccgccgtg 540
ctgcagagca gcggcctgta cagcctgagc agcgtggtga ccgtgcccag cagcagcctg 600
ggcacccaga cctacatctg caacgtgaac cacaagccca gcaacaccaa ggtggacaag 660
agggtggagc ccaagagctg cgacaagacc cacacctgcc ccccctgccc cgcccccgag 720
ctgctgggcg gccccagcgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 780
agcaggaccc ccgaggtgac ctgcgtggtg gtggacgtga gccacgagga ccccgaggtg 840
aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gcccagggag 900
gagcagtaca acagcaccta cagggtggtg agcgtgctga ccgtgctgca ccaggactgg 960
ctgaacggca aggagtacaa gtgcaaggtg agcaacaagg ccctgcccgc ccccatcgag 1020
aagaccatca gcaaggccaa gggccagccc agggagcccc aggtgtacac cctgcccccc 1080
agcagggagg agatgaccaa gaaccaggtg agcctgacct gcctggtgaa gggcttctac 1140
cccagcgaca tcgccgtgga gtgggagagc aacggccagc ccgagaacaa ctacaagacc 1200
accccccccg tgctggacag cgacggcagc ttcttcctgt acagcaagct gaccgtggac 1260
aagagcaggt ggcagcaggg caacgtgttc agctgcagcg tgatgcacga ggccctgcac 1320
aaccactaca cccagaagag cctgagcctg agccccggc 1359
<210> 2
<211> 453
<212> PRT
<213>heavy chain (heavy chain of Aducanumab) of Aducanumab
<400> 2
Met Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Phe Asp Gly Thr Lys Lys Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Arg Gly Ile Gly Ala Arg Arg Gly Pro Tyr Tyr Met Asp
100 105 110
Val Trp Gly Lys Gly Thr Thr 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 Arg 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
450
<210> 3
<211> 645
<212> DNA
<213>light chain (light chain of Aducanumab) of Aducanumab
<400> 3
atggacatcc agatgaccca gagccccagc agcctgagcg ccagcgtggg cgacagggtg 60
accatcacct gcagggccag ccagagcatc agcagctacc tgaactggta ccagcagaag 120
cccggcaagg cccccaagct gctgatctac gccgccagca gcctgcagag cggcgtgccc 180
agcaggttca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctgcag 240
cccgaggact tcgccaccta ctactgccag cagagctaca gcacccccct gaccttcggc 300
ggcggcacca aggtggagat caagaggacc gtggccgccc ccagcgtgtt catcttcccc 360
cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tgtgcctgct gaacaacttc 420
taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc 480
caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg 540
accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 600
ggcctgagca gccccgtgac caagagcttc aacaggggcg agtgc 645
<210> 4
<211> 215
<212> PRT
<213>light chain (light chain of Aducanumab) of Aducanumab
<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 Arg Ala Ser Gln Ser Ile Ser Ser
20 25 30
Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ala Ala Ser Ser Leu Gln 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 Ser Tyr Ser Thr Pro
85 90 95
Leu Thr Phe Gly Gly 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
210 215
Claims (9)
1. application of the plant as host in expression Aducanumab antibody；The plant is selected from romaine lettuce, spinach, tomato, trailing plants
Fore-telling, Chinese cabbage, corn and soybean, wheat or tobacco；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 Aducanumab antibody.
3. expression vector according to claim 2, which is characterized in that the sequence of heavy chain of the Aducanumab antibody is light
Chain-ordering is that the codon optimization of the light chain by the heavy chain of Aducanumab antibody, Aducanumab antibody is the close of favorite plant
Numeral, the sequence of heavy chain of the optimization Aducanumab antibody of acquisition or the Aducanumab antibody light chain sequences of optimization.
4. expression vector according to claim 3, which is characterized in that the heavy chain of the Aducanumab antibody of the optimization
Amino acid sequence is as shown in SEQ ID No.2；The nucleotide sequence such as SEQ of the heavy chain of the Aducanumab antibody of the optimization
Shown in ID No.1；
The amino acid sequence of the light chain of the Aducanumab antibody of the optimization is as shown in SEQ ID No.4；The optimization
The nucleotide sequence of the light chain of Aducanumab antibody is as shown in SEQ ID No.3.
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: being respectively favorite plant by the codon optimization of Aducanumab heavy chain of antibody, Aducanumab antibody light chain
Codon obtains:
The sequence of heavy chain of the Aducanumab antibody of I optimization；
The sequence of light chain of the Aducanumab antibody of II optimization；
Step 2: being separately added into the restricted digestion of Xbal in 5 ' ends of the sequence of heavy chain of the Aducanumab antibody of the optimization
Site is separately added into Sac I site in 3 ' ends；
Xbal restriction enzyme site is added in 5 ' ends of the sequence of light chain of the Aducanumab antibody of the optimization, at 3 ' ends
Sac I site is added in end；
It is cloned into pUC57 carrier, obtains pAduca-H, pAduca-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-Aduca-H, p35S-Aduca-L respectively.
7. according to application of the described in any item expression vectors of claim 2 to 6 in expression Aducanumab antibody.
8. a kind of method of plant as host expresses Aducanumab antibody, which is characterized in that will be such as claim 2 to 6 times
Expression vector described in one is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing
Protein obtains Aducanumab 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.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114457027A (en) * | 2020-10-30 | 2022-05-10 | 未来智人再生医学研究院(广州)有限公司 | Pluripotent stem cell expressing Amyloid beta antibody, derivative and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102762220A (en) * | 2007-10-15 | 2012-10-31 | 森托科尔奥索生物科技公司 | Human anti-amyloid antibodies, compositions,methods and uses |
| CN109476730A (en) * | 2016-06-07 | 2019-03-15 | 生物基因国际神经科学有限责任公司 | The method for treating Alzheimer disease |
| CN109777824A (en) * | 2019-02-21 | 2019-05-21 | 王跃驹 | Application of the plant as host in expression HIV neutralizing antibody |
-
2019
- 2019-08-13 CN CN201910743916.9A patent/CN110511276A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102762220A (en) * | 2007-10-15 | 2012-10-31 | 森托科尔奥索生物科技公司 | Human anti-amyloid antibodies, compositions,methods and uses |
| CN109476730A (en) * | 2016-06-07 | 2019-03-15 | 生物基因国际神经科学有限责任公司 | The method for treating Alzheimer disease |
| CN109777824A (en) * | 2019-02-21 | 2019-05-21 | 王跃驹 | Application of the plant as host in expression HIV neutralizing antibody |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114457027A (en) * | 2020-10-30 | 2022-05-10 | 未来智人再生医学研究院(广州)有限公司 | Pluripotent stem cell expressing Amyloid beta antibody, derivative and application thereof |
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Application publication date: 20191129 |