CN107254486A - Application of the romaine lettuce as host in expression growth factor - Google Patents
Application of the romaine lettuce as host in expression growth factor Download PDFInfo
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- CN107254486A CN107254486A CN201710458332.8A CN201710458332A CN107254486A CN 107254486 A CN107254486 A CN 107254486A CN 201710458332 A CN201710458332 A CN 201710458332A CN 107254486 A CN107254486 A CN 107254486A
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- fgf
- growth factor
- romaine lettuce
- expression
- expression vector
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- 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
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Abstract
The present invention relates to biological technical field, more particularly to application of the romaine lettuce as host in expression growth factor.The present invention expresses human acid fibroblast growth factor (FGF 1) and basic fibroblast growth factor (FGF 2) using recombinant vector and agriculture bacillus mediated vacuum infiltration methods.The expression system determines that plant foreign protein can just be collected after Agrobacterium infects 4d.Restructuring FGF 1 and the successful expressions of FGF 2 are determined using SDS PAGE methods, Western blot (Western methods).Cell proliferation experiment proves that the FGF 1 and FGF 2 of romaine lettuce production have biological activity.The invention provides a kind of low cost, the method for conveniently, largely producing active recombined human FGF 1 and FGF 2.
Description
Technical field
The present invention relates to biological technical field, more particularly to application of the romaine lettuce as host in expression growth factor.
Background technology
Fibroblast growth factor (FGFs) includes the large gene family for participating in growth and differentiation.In invertebrate and
Find to find fibroblast growth factor in vertebrate.Verified FGF is in the development of various tissues and organ, metabolism
Played an important role with reparation.Human fibroblastic growth factor is initially identified as that fibroblast proliferation can be promoted
Protein, it is now known that include 22 members.Due to its potential biological function, FGF has been used for regenerating damaged tissue,
Including skin, blood vessel, muscle, fat, tendon/ligament, cartilage, bone, tooth and nerve.Then, the FGF of regeneration prospect
Source is used together with recombined human FGF families.In fact, FGFs is directly applied to wound location by many previous studies, with
Promote wound healing.
Studied in fibroblast growth factor extended familys widest two members be acidic fibroblast growth because
Sub (FGF-1) and basic fibroblast growth factor (FGF-2).FGF-1 is also referred to as endothelial growth factor, is to have silk
Divide the member of the FGF families of peptide, it is at present by the protein group of at least seven kinds display 35-55% conserved amino acid sequences
Into.Different from other family members, FGF-1 and FGF-2 lack signal peptide, and by addition to classical protein secretion pathway
Mechanism apparently secrete.FGF-1 is largely detected in the brain.It is thin that known expression FGF-1 other cells include liver
Born of the same parents, vascular smooth muscle cells, CNS neurons, Skeletal Muscle Cell, fibroblast, keratinocyte, endothelial cell, intestines post
Columnar epithelium cell and hypophysis basophilic granulocyte and eosinophil.As other FGF, FGF-1 shows sizable
Cross-species reactivity.FGF-2 can be by endothelial cell, smooth muscle cell, macrophages secrete.Its effect is to promote endothelium
The migration of cell and the propagation of smooth muscle cell, it is impossible to make smooth muscle cell migration.Neovascularization can be promoted, infringement is repaired
Endothelial cell.FGF1 and FGF-2 stimulates the propagation of all cells of mesoderm origin, and many neuroderms, outer embryo
The cell of layer and endodermal origin.
Fibroblast growth factor is powerful, with deep layer repair, then current clinical medicine and surgical operation
With play immeasurable great function in beauty treatment, and it is new quickly to repair burn, scratch, scald, tumble injury etc.
The fresh surface of a wound;But the half-life short (2min) of fibroblast growth factor in vivo, and be easily easily degraded by proteases.FGF
Limited availability make it difficult to meet wilderness demand internal and external needed for application.Therefore, for how to reduce experiment and face
The restructuring FGF of bed application cost and high expression FGF production requirement are increasing.In the past few decades, including restructuring gland
Correlated virus (rAAV), Escherichia coli, pichia pastoris phaff insect cell, mammalian cell, baculoviral and transgenosis
Several expression systems including plant attempt to produce restructuring FGF.However, it is insoluble, yield poorly, complicated processing and low bioactivity
The limitation of increasing cell therapy and translation medical application is particularly subject to when meeting the market demand, seriously limiting it should
With.
Expression and production system of the plant as pharmaceutical protein, have studied nearly 30 years.Except low cost and production
Outside the high advantage of amount, the expression system based on plant also reduces humans and animals pathogen and passed during produce protein
It is multicast to the risk of the mankind.In addition, plant eukaryotic albumen inner membrance expression system and secretory pathway are similar to mammalian cell.Plant
Expression system can largely produce macromolecule and multi-subunit pharmaceutical protein, and be significantly better than prokaryotic expression system,
Such as escherichia expression system.If desired for posttranslational modification, glycosylated albumen, and need the monoclonal assembled to resist
Body, it can not be realized with prokaryotic system.Pharmaceutical protein by the use of plant production has been commercialized as biological reagent, or uses writer
The vaccine additive of fowl.2012, food and drug administration (FDA) have approved to be thanked for treating the type dagger-axe of genetic disease 1
The albumen ELELYSO of diseaseTM(taliglucerase alfa), and this albumen is produced using carrot.Past 10 years
In, people sharply increase to the demand of pharmaceutical protein, so FDA ratifies the number of the plant medical protein for clinical test
Amount is also being continuously increased.
Plant transient expression system, which can largely produce recombinant protein, is used for clinical research or reply sudden illness.
2014, it is uniquely used for being effective against the Antybody therapy medicine of Ebola virus outburst, ZMappTM is exactly Agrobacterium infiltration side
Method is produced in tobacco leaf.ZMapp effect and security opens road for the industry of promotion plant pharmacy industry.At present,
The most common host plant of tobacco moment protein expression, and various carriers and Agrobacterium permeating method have been developed, it is used for
Mass produced in a short time.However, tobacco has high microsteping content and potential toxic compounds, such as alkaloid Buddhist nun
Gu Ding, significantly increases the cost in downstream purification process, greatly hinders the further development of plant foreign protein medicine.
Compared with tobacco leaf system, less phenols and toxic compounds are contained in romaine lettuce, therefore, is grown romaine lettuce as host in expression
The factor has important practical significance.
The content of the invention
In view of this, the present invention provides application of the romaine lettuce as host in expression growth factor.The present invention utilizes romaine lettuce
The active platform produced as recombinant protein, eliminates plant growing cycle, greatlys save the time that early stage cultivates plant.This
Invention romaine lettuce system expression FGF-1 and FGF-2, and active external source egg is successfully separated out under mild conditions
In vain, it was demonstrated that romaine lettuce expression platform can be for production FGF fibroblast growth factors.
In order to realize foregoing invention purpose, the present invention provides following technical scheme:
Application the invention provides romaine lettuce as host in expression growth factor.
In some specific embodiments of the present invention, the growth factor is selected from FGF-1 or FGF-2.
Present invention also offers the nucleotide sequence and binary plant carrier of a kind of expression vector, including growth factor.
The present invention some specific embodiments in, growth factor described in the expression vector include FGF-1 or
FGF-2。
In some specific embodiments of the present invention, the construction method of the expression vector comprises the following steps:
Step 1:Xbal restriction enzyme sites and Kpnl are separately added into 5 ' ends of the nucleotide sequence of growth factor
Restriction enzyme site, Sacl restriction enzyme sites and Pacl restriction enzyme sites are separately added into 3 ' ends;The life
The long factor includes FGF-1 or FGF-2;
Step 2:Cloned by ThermoFisher, respectively obtain 17ABRZ5P_FGF1_pMA-T cloning vectors or
17ABRZ4P_FGF2_pMA-T cloning vectors;
Step 3:As Kpnl/Sacl respectively from step 2 obtained by cloning vector in obtain genetic fragment FGF-1 or FGF-
2, binary plant carrier pCam35S is cloned into, expression vector-FGF1 or-FGF2 is obtained respectively.
Specifically, the construction method for the expression vector that the present invention is provided is:By people FGF-1 (GenBank Accession
Number:) and FGF-2 (GenBank Accession NM_001257210.1:M27968.1) codon optimization is favorite plant
Codon, by GeneArtTMGeneOptimizerTM(ThermoFisher) design and synthesize.Optimization FGF-1 and
The end of FGF-2 sequences 5 ' adds Xbal and Kpnl restriction enzyme sites, in 3 ' ends addition Sacl and Pacl sites, and by
ThermoFisher is cloned into 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T carriers.Growth factor base
Because of fragment FGF-1 and FGF-2 by Kpnl/Sacl from 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T
Middle separation, and binary plant carrier pCam35S is cloned into, transient expression vector p35S-FGF1 and p35S- are produced respectively
FGF2.By two kinds of plant expression constructs respectively by using Multiporator (Eppendorf, Hamburg, Germany) electricity
Perforation is transformed into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over containing kanamycins antibiotic (50mg/
L on selective LB flat boards).28 DEG C are incubated after 2d in the dark, and picking single bacterium colony is inoculated into 0.5L YEB (yeast extract meat
Soup, 5g/L sucrose, 5g/L tryptones, 6g/L yeast extracts, 0.24g/L MgSO4, pH7.2) and supplement antibiotic liquid
Culture medium (50mg/L kanamycins).The culture of inoculation is incubated 72h in oscillator (220rpm) with 25~28 DEG C.Pass through
Addition YEB culture mediums measurement OD600 values are simultaneously adjusted to 3.5~4.5.Then nutrient solution is collected, (4500 rotating speed) 10min is centrifuged.
Agrobatcerium cell is resuspended in osmotic medium (10mM MES, 10mM MgSO4) in O.D.600 be 0.5.The clone
FGF-1 and FGF-2 genetic fragments (Figure 1A, B), and build two kinds based on binary plant expression vector p35S-FGF1 and
P35S-FGF2 (Fig. 2).After construct is completed, confirm that genetic fragment is complete with specific restriction enzyme digestion.
In the present invention, FGF-1cDNA sequences (GenBank:NM_001257210.1) as shown in SEQ ID No.1;
FGF-1 amino acid sequences are as shown in SEQ ID No.2;FGF-2cDNA sequences (GenBank:M27968.1) such as SEQ ID No.3
It is shown;FGF-2 amino acid sequences are as shown in SEQ ID No.4;FGF-1cDNA sequences such as SEQ ID after codon optimization
Shown in No.5;FGF-2cDNA sequences after codon optimization are as shown in SEQ ID No.6.
Present invention also offers application of the expression vector in expression growth factor.Some in the present invention are specific real
Apply in scheme, the growth factor includes FGF-1 or FGF-2.
Present invention also offers a kind of method of romaine lettuce as host expresses growth factor, described expression vector is converted
Into Agrobacterium, entered by agriculture bacillus mediated vacuum infiltration after romaine lettuce tissue, extracting and developing protein, obtain growth factor.
In some specific embodiments of the present invention, growth factor described in methods described includes FGF-1 or FGF-2.
In some specific embodiments of the present invention, the agriculture bacillus mediated vacuum infiltration comprises the following steps:
Step 1:Vacuumize 25~45s;
Step 2:Keep 30~60s of vacuum (- 95kPa) pressure;
Step 3:Release pressure causes penetrating fluid to penetrate into the plant tissue;
Repeat the above steps 2~3 times, lucifuge processing 4d.
In some specific embodiments of the present invention, the Agrobacterium is Agrobacterium tumefaciens GV3101.
Specifically, the method for agriculture bacillus mediated vacuum infiltration is:The Agrobacterium culture suspension prepared is placed in into 2L to burn
Cup, is placed in drier.The romaine lettuce that this laboratory is preserved is inverted (core is upward) and lightly rotates on bacterial suspension
In, drier is sealed.Vavuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visual penetration liquid
In leaf tissue.Keep 30~60s of pressure state.Quickly open the system to discharge pressure, penetrating fluid is penetrated into tissue
Space.The process is repeated 2~3 times, until high-visible penetrating fluid spreads substantially in romaine lettuce tissue.Then by romaine lettuce tissue
Gently taken out from penetrating fluid, and with distilled water continuous flushing three times, in the container for being then transferred into plastic foil covering.Will processing
Sample keep 4d in the dark.
After infiltration, most romaine lettuce flood during being organized in vacuum immersion, in addition to firm middle rib region, remaining
Part shows khaki region after 4 days in vacuum infiltration.In order to increase the quantity for the soil Agrobacterium for immersing leaf texture, make
From the beginning 10% romaine lettuce is cut away with scissors so that romaine lettuce leaf texture is infiltrated in penetrating fluid as far as possible, and is discharged.With it is longer
Vacuum open-assembly time compare, the method reduce leaf tissue necrosis.
In some specific embodiments of the present invention, extracting and developing protein is specially:
Romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with agitator, and is 1 with volume ratio:The extraction buffering of 1 ratio
Liquid (100mM KPi, pH7.8;5mM EDTA;10m- mercaptoethanol) 1~2min of mixer high speed homogenate.By homogenate
Regulation is 8.0 to pH, and with filtered through gauze, filtrate with 10,000g centrifuges 15min to remove cell fragment at 4 DEG C.Collect supernatant
Liquid, is mixed with ammonium sulfate (50%), and is incubated 60min shaking on ice.Divided again at 4 DEG C by centrifuge (10,000g)
From 15min.Obtained supernatant is carried out second and takes turns ammonium sulfate (70%) precipitation, suspension 60min is shaken on ice, again at 4 DEG C
Under with 10,000g centrifuge 15min.Then, abandoning supernatant, 5mL buffer solutions (20mM is dissolved in by processing sample pellet protein
KPi, pH 7.8;2mM EDTA;10m- mercaptoethanol) in and at 4 DEG C store.
The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) loadings of sample (5 ì L) thermal denaturation are taken
Buffer solution (Biorad, Hercules, CA, USA) is 4~12%Bis-Tris Plus SDS- gels
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis, is then dyed with Coomassie blue G250 (Biorad)
Gel is taken pictures again afterwards.Western Blot western blots hybridization for recombinating FGF-1 and FGF-2,10 ì l's
Sample and hFGF-1 and hFGF-2 standard items (Biovision) are recombinated respectively 10~20%Bis-Tris Plus
Separated on polyacrylamide gel, and by its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, with anti-hFGF-1 and
HFGF-2 antibody (Abcam) carries out immune response, dilution 1 respectively:10000 and horseradish peroxidase (HRP) mark mountain
Goat anti-rabbit igg (Beyotime), dilution factor is respectively 1:20000, and use ECL plus (Amersham Biosciences)
Show, display image is taken pictures.
The Downstream processing of the recombinant protein of plant origin is typically difficult to and expensive, because cellulose cell wall is difficult to split
Solution and secondary plant metabolites.The present invention is stirred with mixer and is homogenized, and greatlys save homogenate cost and technique.Restructuring
FGF-1 and FGF-2 separates us by SDS-PAGE and observes that estimation molecular weight is about 17kDa band in swimming lane
(Fig. 3 A), without significantly corresponding band in stealth control swimming lane.Compareed based on Bradford determination methods and spectrodensitometry
The protein content that group determines purification of samples is 0.58mg/g.In addition, Western blot analysis also detects that about 17kDa bar
Band (Fig. 3 B), it was observed that molecular weight of albumen (17kDa) it is consistent with positive controls.
Containing 10% (v/v) hyclone (FBS, Gibco) from MEC NIH/3T3 cell lines
Dulbecco improvement Eagle culture mediums (DMEM, Gibco) in 37 DEG C, in 5%CO2Middle culture.By NIH/3T3 cells
100% is grown in 24 orifice plates to converge.Serum starved cells 16h, with sterile liquid relief syringe needle scratch NIH/3T3 surfaces, uses PBS
Washing removes cell fragment.Then the FGF-1 and FGF-2 and standard items purified with 100ng/ml dosage stimulate cell
48h.Region is being scratched to initial wound and cell using microscope (Nikon).
The haFGF of purifying bioactivity is studied by cell experiment.First, using purifying restructuring FGF-1 and
FGF-2 cultivates NIH/3T3 cells, and positive control is used as with FGF-1 and FGF-2 standards using identical business.NIH/3T3's
Cell propagation can by dosage by using 100ng/ml dosage purification of Recombinant FGF-1 and FGF-2 when find, in 48h
Time point check cell growth result show that the NIH/3T3 cell growths without any processing are poor.By contrast, use
The restructuring FGF-1 and FGF-2haFGF of the purifying or NIH/3T3 of equal positive control FGF-1 and FGF-2 standard processing
Cell growth is good;They spread (Fig. 4) in scratch surface stretching.These results indicate that passing through romaine lettuce system Transient Expression
External source FGF-1 and FGF-2 have biological activity, and romaine lettuce system is probably the batch production of bioactivity recombinant pharmaceutical proteins matter
Suitable bioreactor.
The growth time of the tobacco plant permeated for vacuum Agrobacterium was usually 4 to 6 weeks.The present invention by the use of romaine lettuce as
The active platform of recombinant protein production, eliminates plant growing cycle, greatlys save the time that early stage cultivates plant.The present invention
The FGF-1 and FGF-2 with romaine lettuce system expression, and it is successfully separated out active foreign protein under mild conditions,
Prove that romaine lettuce expression platform can be for production FGF fibroblast growth factors.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
There is the accompanying drawing used required in technology description to be briefly described.
Fig. 1 (A) shows FGF-1 and FGF-2 cloning vectors (ThermoFisher builds synthesis);
Fig. 1 (B) shows that FGF-1 and FGF-2 genetic fragments digestion (KpnI/SacI) is identified;
Fig. 2 shows that plant transient expression vector p35S-FGF1 and p35S-FGF2 build flow;Utilize restriction enzyme
(KpnI/SacI) double digestion, cuts FGF-1 and FGF-2 fragments from Fig. 1 cloning vectors, connects into pCam35S's respectively
KpnI/SacI sites, generation plant transient expression vector p35S-FGF1 and p35S-FGF2;
Wherein, 35S, with ' the UTR CaMV 35S promoters of tobacco mosaic virus (TMV) (TMV) 5;NPT II, for card, that is mould
The expression of the coding nptII genes of plain resistance;Nos 3 ', terminator;
Fig. 3 (A) shows the restructuring fibroblastic growth using polyacrylamide gel electrophoresis (SDS-PAGE) detection purifying
The factor;Swimming lane 1:Purification of Recombinant FGF-1 (5 μ g);Swimming lane 2:Purification of Recombinant FGF-2 (5 μ g);Swimming lane 3:Positive control FGF-1 (5 μ
g);Swimming lane 4:Positive control FGF-2 (5 μ g);Swimming lane 5:Antivacuum infiltration blade eluent negative control;
Fig. 3 (B) shows the restructuring fibroblast growth factor of Western markings hybridization check purifying;Swimming lane 1:Purifying weight
Group FGF-1 (5 μ g);Swimming lane 2:Purification of Recombinant FGF-2 (5 μ g);3:Positive control FGF-1 (5 μ g);Swimming lane 4:Positive control FGF-
2(5μg);Swimming lane 5:Antivacuum infiltration blade eluent negative control;
Fig. 4 shows that cell proliferation is tested;Cut NIH/3T3 cells are handled using the FGF-1 and FGF-2 of purifying, are healed
Determine;After 48 hours, restructuring fibroblast growth factor is obviously promoted cell-proliferation activity.
Embodiment
Application the invention discloses romaine lettuce as host in expression growth factor, those skilled in the art can use for reference
Present disclosure, is suitably modified technological parameter realization.In particular, all similar replacements and change are to this area skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.The present invention method and application by compared with
Good embodiment is described, and related personnel substantially can be not departing from present invention, in spirit and scope to as described herein
Methods and applications are modified or suitably change is with combining, to realize and apply the technology of the present invention.
Present invention research shows that romaine lettuce system can be effectively expressing platform, be quick, transient expression recombinant protein
There is provided method.Vacuum Agrobacterium permeating method is simple, quickly, reduces blade necrosis, and can improve recombinant protein yield.
Romaine lettuce can increase protein output by bearing vacuum pressure, and allow the more complete infiltration of every leaf.Due to romaine lettuce
Be easy to growth and can commercial a large amount of productions, therefore compare other transient expression plants, such as tobacco be easier acquisition and more
Cheaply.And due to not needing special installation or liquid nitrogen, cost benefit is higher.Present invention demonstrates that this method can be used for the short time
Interior large-scale production FGF-1 and FGF-2 recombinant proteins.
Can be by as raw materials used and reagent in application of the host in expression growth factor the invention provides romaine lettuce
Market is bought.
With reference to embodiment, the present invention is expanded on further:
The structure of the plant transient expression vector of embodiment 1
It is of the invention by people FGF-1 (GenBank Accession in order to provide high efficient expression of the foreign protein in plant
Number:) and FGF-2 (No. GenBankAccession NM_001257210.1:M27968.1) codon optimization is favorite plant
Codon, by GeneArtTMGeneOptimizerTM(ThermoFisher) design and synthesize.Optimization FGF-1 and
The end of FGF-2 sequences 5 ' adds Xbal and Kpnl restriction enzyme sites, in 3 ' ends addition Sacl and Pacl sites, and by
ThermoFisher is cloned into 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T carriers.Growth factor base
Because of fragment FGF-1 and FGF-2 by Kpnl/Sacl from 17ABRZ5P_FGF1_pMA-T and 17ABRZ4P_FGF2_pMA-T
Middle separation, and binary plant carrier pCam35S is cloned into, transient expression vector p35S-FGF1 and p35S- are produced respectively
FGF2.By two kinds of plant expression constructs respectively by using Multiporator (Eppendorf, Hamburg, Germany) electricity
Perforation is transformed into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over containing kanamycins antibiotic (50mg/
L on selective LB flat boards).28 DEG C are incubated after 2d in the dark, and picking single bacterium colony is inoculated into 0.5L YEB (yeast extract meat
Soup, 5g/L sucrose, 5g/L tryptones, 6g/L yeast extracts, 0.24g/L MgSO4, pH7.2) and supplement antibiotic liquid
Culture medium (50mg/L kanamycins).The culture of inoculation is incubated 72h in oscillator (220rpm) with 25~28 DEG C.Pass through
Addition YEB culture mediums measurement OD600 values are simultaneously adjusted to 3.5~4.5.Then nutrient solution is collected, (4500 rotating speed) 10min is centrifuged.
Agrobatcerium cell is resuspended in osmotic medium (10mM MES, 10mM MgSO4) in O.D.600 be 0.5.
Clone's FGF-1 and FGF-2 genetic fragments (Figure 1A, B), and build two kinds of plants based on dual factors virus
Thing expression vector p35S-FGF1 and p35S-FGF2 (Fig. 2).After construct is completed, gene is confirmed with specific restriction enzyme digestion
Fragment is complete.
The agriculture bacillus mediated vacuum infiltration of embodiment 2
Present invention optimizes the method for Agrobacterium vacuum infiltration (Fig. 2).The Agrobacterium culture suspension prepared is placed in
2L beakers, are placed in drier.The romaine lettuce that this laboratory is preserved, which is inverted (core is upward) and lightly rotates on bacterium, to be hanged
In supernatant liquid, drier is sealed.Vavuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visible oozed
Transparent liquid is in leaf tissue.Keep 30~60s of pressure state.Quickly open the system to discharge pressure, make penetrating fluid infiltration group
Knit interior space.The process is repeated 2~3 times, until high-visible penetrating fluid spreads substantially in romaine lettuce tissue.Then by romaine lettuce
Tissue gently takes out from penetrating fluid, and with distilled water continuous flushing three times, is then transferred into the container that plastic foil is covered.Will
The sample of processing keeps 4d in the dark.
After infiltration, most romaine lettuce flood during being organized in vacuum immersion, in addition to firm middle rib region, remaining
Part shows khaki region after 4 days in vacuum infiltration.In order to increase the quantity for the soil Agrobacterium for immersing leaf texture, make
From the beginning 10% romaine lettuce is cut away with scissors so that romaine lettuce leaf texture is infiltrated in penetrating fluid as far as possible, and is discharged.With it is longer
Vacuum open-assembly time compare, the method reduce leaf tissue necrosis.
The Protein Extraction of embodiment 3 and separation
Romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with agitator, and is 1 with volume ratio:The extraction buffering of 1 ratio
Liquid (100mM KPi, pH7.8;5mM EDTA;10m- mercaptoethanol) 1~2min of mixer high speed homogenate.By homogenate
Regulation is 8.0 to pH, and with filtered through gauze, filtrate with 10,000g centrifuges 15min to remove cell fragment at 4 DEG C.Collect supernatant
Liquid, is mixed with ammonium sulfate (50%), and is incubated 60 minutes shaking on ice.Divided again at 4 DEG C by centrifuge (10,000g)
From 15min.Obtained supernatant is carried out second and takes turns ammonium sulfate (70%) precipitation, suspension 60min is shaken on ice, again at 4 DEG C
Under with 10,000g centrifuge 15min.Then, abandoning supernatant, 5mL buffer solutions (20mM is dissolved in by processing sample pellet protein
KPi, pH 7.8;2mM EDTA;- mercaptoethanol) in and at 4 DEG C store.
The PAGE gel electrophoresis of embodiment 4 and the hybridization of Western Blot western blots
The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) loadings of sample (5 ì L) thermal denaturation are taken
Buffer solution (Biorad, Hercules, CA, USA) is 4~12%Bis-Tris Plus SDS- gels
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis, is then dyed with Coomassie blue G250 (Biorad)
Gel is taken pictures again afterwards.Western Blot western blots hybridization for recombinating FGF-1 and FGF-2,10 ì l's
Sample and hFGF-1 and hFGF-2 standard items (Biovision) are recombinated respectively 10~20%Bis-Tris Plus
Separated on polyacrylamide gel, and by its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, with anti-hFGF-1 and
HFGF-2 antibody (Abcam) carries out immune response, dilution 1 respectively:10000 and horseradish peroxidase (HRP) mark mountain
Goat anti-rabbit igg (Beyotime), dilution factor is respectively 1:20000, and use ECL plus (Amersham Biosciences)
Show, display image is taken pictures.
The Downstream processing of the recombinant protein of plant origin is typically difficult to and expensive, because cellulose cell wall is difficult to split
Solution and secondary plant metabolites.We are stirred with mixer and are homogenized, and greatly save homogenate cost and technique.Recombinate FGF-
1 and FGF-2 separates us by SDS-PAGE and observes that estimation molecular weight is about 17kDa band (figure in swimming lane
3A), without significantly corresponding band in stealth control swimming lane.Surveyed based on Bradford determination methods and spectrodensitometry control group
The protein content for determining purification of samples is 0.58mg/g.In addition, Western blot analysis also detects that about 17kDa band (figure
3B), it was observed that molecular weight of albumen (17kDa) it is consistent with positive controls.
The cell proliferation of embodiment 5 is tested
Containing 10% (v/v) hyclone (FBS, Gibco) from MEC NIH/3T3 cell lines
Dulbecco improvement Eagle culture mediums (DMEM, Gibco) in 37 DEG C, in 5%CO2Middle culture.By NIH/3T3 cells
100% is grown in 24 orifice plates to converge.Serum starved cells 16h, with sterile liquid relief syringe needle scratch NIH/3T3 surfaces, uses PBS
Washing removes cell fragment.Then the FGF-1 and FGF-2 and standard items purified with 100ng/ml dosage stimulate cell
48h.Region is being scratched to initial wound and cell using microscope (Nikon).
The haFGF of purifying bioactivity is studied by cell experiment.First, using purifying restructuring FGF-1 and
FGF-2 cultivates NIH/3T3 cells, and positive control is used as with FGF-1 and FGF-2 standards using identical business.NIH/3T3's
Cell propagation can by dosage by using 100ng/ml dosage purification of Recombinant FGF-1 and FGF-2 when find, in 48h
Time point check cell growth result show that the NIH/3T3 cell growths without any processing are poor.By contrast, use
The restructuring FGF-1 and FGF-2haFGF of the purifying or NIH/3T3 of equal positive control FGF-1 and FGF-2 standard processing
Cell growth is good;They spread (Fig. 4) in scratch surface stretching.These results indicate that passing through romaine lettuce system Transient Expression
External source FGF-1 and FGF-2 have biological activity, and romaine lettuce system is probably the batch production of bioactivity recombinant pharmaceutical proteins matter
Suitable bioreactor.
Embodiment 6
Control group:Utilize leaf tobacco production PD-1 and PD-L1 antibody;
Experimental group:The romaine lettuce production PD-1 and PD-L1 antibody that the present invention is provided;
The people of table 1 acidity and basic fibroblast growth factor
*Show P≤0.05 compared with control group;#Show P≤0.01 compared with control group;
As shown in Table 1, compared with the tobacco leaf system of control group, romaine lettuce transient expression human acid fibroblast growth factor
(FGF-1) and basic fibroblast growth factor (FGF-2), notable (P≤0.05) shortens the production cycle, significantly (P≤
0.05) protein content is improved, notable (P≤0.05) improves protein active, simplifies the complexity of protein purification, extremely shown
Write (P≤0.01) and reduce production cost.
Summary result of the test shows that botanical system especially romaine lettuce system is more economical, efficiently expresses platform.
Can quick transient expression recombinant protein, can mass produce that people is acid and basic fibroblast life in a short time
The long factor.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>Shenzhen Hui Sheng bio tech ltd
<120>Application of the romaine lettuce as host in expression growth factor
<130> MP1710873
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 468
<212> DNA
<213> FGF-1
<400> 1
atggctgaag gggaaatcac caccttcaca gccctgaccg agaagtttaa tctgcctcca 60
gggaattaca agaagcccaa actcctctac tgtagcaacg ggggccactt cctgaggatc 120
cttccggatg gcacagtgga tgggacaagg gacaggagcg accagcacat tcagctgcag 180
ctcagtgcgg aaagcgtggg ggaggtgtat ataaagagta ccgagactgg ccagtacttg 240
gccatggaca ccgacgggct tttatacggc tcacagacac caaatgagga atgtttgttc 300
ctggaaaggc tggaggagaa ccattacaac acctatatat ccaagaagca tgcagagaag 360
aattggtttg ttggcctcaa gaagaatggg agctgcaaac gcggtcctcg gactcactat 420
ggccagaaag caatcttgtt tctccccctg ccagtctctt ctgattga 468
<210> 2
<211> 155
<212> PRT
<213> FGF-1
<400> 2
Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe
1 5 10 15
Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser
20 25 30
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly
35 40 45
Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu
50 55 60
Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
65 70 75 80
Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu
85 90 95
Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr
100 105 110
Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
115 120 125
Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala
130 135 140
Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp
145 150 155
<210> 3
<211> 468
<212> DNA
<213> FGF-2
<400> 3
atggcagccg ggagcatcac cacgctgccc gccttgcccg aggatggcgg cagcggcgcc 60
ttcccgcccg gccacttcaa ggaccccaag cggctgtact gcaaaaacgg gggcttcttc 120
ctgcgcatcc accccgacgg ccgagttgac ggggtccggg agaagagcga ccctcacatc 180
aagctacaac ttcaagcaga agagagagga gttgtgtcta tcaaaggagt gtgtgctaac 240
cgttacctgg ctatgaagga agatggaaga ttactggctt ctaaatgtgt tacggatgag 300
tgtttctttt ttgaacgatt ggaatctaat aactacaata cttaccggtc aaggaaatac 360
accagttggt atgtggcact gaaacgaact gggcagtata aacttggatc caaaacagga 420
cctgggcaga aagctatact ttttcttcca atgtctgcta agagctga 468
<210> 4
<211> 155
<212> PRT
<213> FGF-2
<400> 4
Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu Asp Gly
1 5 10 15
Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu
20 25 30
Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro Asp Gly Arg
35 40 45
Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys Leu Gln Leu
50 55 60
Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val Cys Ala Asn
65 70 75 80
Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser Lys Cys
85 90 95
Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr
100 105 110
Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp Tyr Val Ala Leu Lys
115 120 125
Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys Thr Gly Pro Gly Gln Lys
130 135 140
Ala Ile Leu Phe Leu Pro Met Ser Ala Lys Ser
145 150 155
<210> 5
<211> 562
<212> DNA
<213>FGF-1 cDNA sequences after codon optimization
<400> 5
tctagaggta ccgtattttt acaacaatta ccaacaacaa caaacaacaa acaacattac 60
aattactatt tacaattaca atggccgagg gcgagatcac cactttcact gctcttaccg 120
agaagttcaa cctgccacca ggcaactaca agaagccaaa gcttctctac tgctccaacg 180
gtggacactt ccttaggatt ctcccagatg gaactgtgga tggcactagg gatagatccg 240
atcagcacat tcagctccag ctgtctgctg agtctgttgg agaggtgtac atcaagtcca 300
ctgagactgg acagtacctc gctatggata ctgatggact cctctacgga tcccagactc 360
caaatgaaga gtgcttgttc cttgagaggc tcgaagagaa ccactacaac acctacatct 420
ccaagaagca cgccgagaag aactggttcg tgggacttaa gaagaacggc tcctgtaaga 480
gaggacccag gactcattat ggccagaagg ccattctgtt cttgcccctt ccagtgtcct 540
ctgactagtt aattaagagc tc 562
<210> 6
<211> 562
<212> DNA
<213>FGF-2 cDNA sequences after codon optimization
<400> 6
tctagaggta ccgtattttt acaacaatta ccaacaacaa caaacaacaa acaacattac 60
aattactatt tacaattaca atggctgccg gctccatcac cactcttcca gctttgcctg 120
aagatggtgg atctggtgct tttccaccag gccatttcaa ggatccaaag aggctctact 180
gcaagaacgg cggattcttc cttaggattc acccagatgg tcgtgtggat ggtgtgcgtg 240
aaaagtccga tccacacatt aagcttcagc tccaggctga agagaggggc gttgtatcta 300
ttaagggcgt gtgcgctaac cgttacctcg ccatgaagga agatggaagg cttctcgctt 360
ctaagtgcgt gacagatgag tgcttcttct tcgaacgact cgaatccaac aactacaaca 420
cctacaggtc ccgtaagtac acctcttggt acgtggcact taagaggacc ggacagtaca 480
agctcggatc taagactgga ccaggacaga aggctatcct cttcttgcca atgtccgcca 540
agtcttagtt aattaagagc tc 562
Claims (10)
1. application of the romaine lettuce as host in expression growth factor.
2. application according to claim 1, it is characterised in that the growth factor is selected from FGF-1 or FGF-2.
3. a kind of expression vector, it is characterised in that nucleotide sequence and binary plant carrier including growth factor.
4. expression vector according to claim 3, it is characterised in that the growth factor includes FGF-1 or FGF-2.
5. the expression vector according to claim 3 or 4, it is characterised in that its construction method comprises the following steps:
Step 1:Xbal restriction enzyme sites and Kpnl limitations are separately added into 5 ' ends of the nucleotide sequence of growth factor
Property restriction enzyme site, Sacl restriction enzyme sites and Pacl restriction enzyme sites are separately added into 3 ' ends;The growth because
Attached bag includes FGF-1 or FGF-2;
Step 2:Cloned by ThermoFisher, 17ABRZ5P_FGF1_pMA-T cloning vectors or 17ABRZ4P_ are obtained respectively
FGF2_pMA-T cloning vectors;
Step 3:As Kpnl/Sacl respectively from step 2 obtained by cloning vector in obtain genetic fragment FGF-1 or FGF-2, gram
It is grand to binary plant carrier pCam35S, expression vector p35S-FGF1 or p35S-FGF2 are obtained respectively.
6. application of the expression vector in expression growth factor according to any one of claim 3 to 5.
7. application according to claim 6, it is characterised in that the growth factor includes FGF-1 or FGF-2.
8. a kind of romaine lettuce is used as the method for host expresses growth factor, it is characterised in that will be such as any one of claim 3 to 5 institute
The expression vector stated is transformed into Agrobacterium, is entered by agriculture bacillus mediated vacuum infiltration after romaine lettuce tissue, extracting and developing albumen
Matter, obtains growth factor.
9. method according to claim 8, it is characterised in that the growth factor includes FGF-1 or FGF-2.
10. method according to claim 8 or claim 9, it is characterised in that the agriculture bacillus mediated vacuum infiltration includes following step
Suddenly:
Step 1:Vacuumize 25~45s;
Step 2:Keep 30~60s of vacuum -95kPa pressure;
Step 3:Release pressure causes penetrating fluid to penetrate into the plant tissue;
Repeat the above steps 2~3 times, lucifuge processing 4d.
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Cited By (8)
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CN107893081A (en) * | 2017-12-27 | 2018-04-10 | 温州大学 | Gene order, expression vector and the production method of a kind of people source keratin cell growth factor 2 |
CN109679986A (en) * | 2017-10-19 | 2019-04-26 | 北京睿诚海汇健康科技有限公司 | Application of the plant as host in expression seven factor of blood coagulation |
CN109679985A (en) * | 2017-10-19 | 2019-04-26 | 北京睿诚海汇健康科技有限公司 | Application of the plant as host in expression nine factor of blood coagulation |
CN110092833A (en) * | 2018-01-30 | 2019-08-06 | 北京睿诚海汇健康科技有限公司 | Application of the plant as host in expression rituximab antibody |
CN110092831A (en) * | 2018-01-30 | 2019-08-06 | 北京睿诚海汇健康科技有限公司 | Application of the plant as host in expression A Damu antibody |
CN110205337A (en) * | 2019-06-24 | 2019-09-06 | 王跃驹 | Application of the plant as host expresses source of people Telomerase |
CN110205338A (en) * | 2019-06-24 | 2019-09-06 | 王跃驹 | Application of the plant as host in expression recombinant human granulocyte colony stimulating factor |
CN111454348A (en) * | 2020-05-22 | 2020-07-28 | 王跃驹 | Method for extracting basic fibroblast growth factor |
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CN107893081A (en) * | 2017-12-27 | 2018-04-10 | 温州大学 | Gene order, expression vector and the production method of a kind of people source keratin cell growth factor 2 |
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CN110205337A (en) * | 2019-06-24 | 2019-09-06 | 王跃驹 | Application of the plant as host expresses source of people Telomerase |
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