CN109456398A - Application of the plant as host in expression epidermal growth factor - Google Patents
Application of the plant as host in expression epidermal growth factor Download PDFInfo
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- CN109456398A CN109456398A CN201710796127.2A CN201710796127A CN109456398A CN 109456398 A CN109456398 A CN 109456398A CN 201710796127 A CN201710796127 A CN 201710796127A CN 109456398 A CN109456398 A CN 109456398A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The present invention relates to field of biotechnology, in particular to application of the plant especially plant as host in expression epidermal growth factor.The present invention expresses human epidermal growth factor (human epidermal growth factor, abbreviation EGF) using recombinant vector and mediated by agriculture bacillus vacuum infiltration methods.The expression system determines that plant foreign protein can be collected after Agrobacterium infects 4d.Recombination EGF successful expression is determined using SDS-PAGE method, Western blot (Western method).Cell proliferation is experiments have shown that the EGF of plant especially romaine lettuce production has biological activity.The present invention provides it is a kind of low cost, conveniently, the method for the active recombined human EGF of mass production.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant is expressing answering in epidermal growth factor as host
With.
Background technique
Growth factor is a kind of naturally occurring material, can be spread, treatment is cured and cell differentiation with stimulating cellular growth.
Growth factor is usually a protein or a steroid hormone.Growth factor has adjusting various kinds of cell process very heavy
The effect wanted.Growth factor promotes cell differentiation and maturation usually as ceS signal molecules.But each growth factor it
Between effect it is different.For example, Bone Morphogenetic Protein stimulates bone cell differentiation, fibroblast growth factor and vascular endothelial growth
The factor stimulates blood vessel differentiation (angiogenesis).
Epidermal growth factor (EGF) is carried out stimulating cellular growth and is divided by being tied to its receptor, EGF-R ELISA
Change.Human epidermal growth factor is a 6kDa protein, there is 53 amino acid residues and three intramolecular disulfide bonds.Recombinant human
Epidermal growth factor, selling brand is under Heberprot-P, for treating diabetic foot ulcer.It can be injected by wound,
Or external application use, evidence its can improve wound healing.
Currently, EGF growth factor produces in Escherichia coli.Expression and production system of the plant as pharmaceutical protein,
It has studied nearly 30 years.Other than in addition to the low cost and yield height etc. the advantages of, the expression system based on plant also reduces people
The risk of the mankind is traveled to during generate protein with animal pathogen.In addition, film expression system in plant eukaryotic albumen
System and secretory pathway are similar to mammalian cell.Plant expression system can mass production macromolecule and multi-subunit drug
Protein, and it is significantly better than prokaryotic expression system, such as escherichia expression system.As needed posttranslational modification, sugar
The albumen of base, and the monoclonal antibody for needing to assemble, cannot be realized with prokaryotic system.Utilize the medicinal egg of plant production
It is white to be commercialized as biological reagent, or the vaccine additive as poultry.2012, food and drug administration
(FDA) the albumen ELELYSO for treating 1 type Gaucher disease of genetic disease is had approvedTM(taliglucerase alfa), and it is this
Albumen is produced using carrot.In the past decade, demand of the people to pharmaceutical protein sharply increases, so FDA ratifies
The quantity of plant medical protein for clinical test is also being continuously increased.
Plant transient expression system can be with mass production recombinant protein for clinical research or reply sudden illness.
It 2014, is uniquely used for being effective against the Antybody therapy drug of Ebola virus outburst, ZMappTM is exactly Agrobacterium infiltration side
Method produces in tobacco leaf.The effect of ZMapp and safety are that the industry of plant pharmacy industry is pushed to open road.Currently,
The most common host plant of tobacco moment protein expression, and various carriers and Agrobacterium permeating method have been developed, it is used for
It is 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 drug.
Compared with tobacco leaf system, less phenols and toxic compounds are contained in romaine lettuce, therefore, using plant especially romaine lettuce as host
It has important practical significance in expression epidermal growth factor.
Summary of the invention
In view of this, the present invention provides application of the plant especially romaine lettuce as host in expression epidermal growth factor.
The active platform that the present invention is produced using romaine lettuce as recombinant protein, eliminates plant growing cycle, and training early period is greatly saved
Educate the time of plant.Present invention romaine lettuce system expression EGF, and be successfully separated out under mild conditions active outer
Source protein, it was demonstrated that romaine lettuce expression platform can be used to produce EGF growth factor.
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 epidermal growth factor；The plant be selected from romaine lettuce,
Chinese cabbage, corn and soybean or wheat；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
In some specific embodiments of the invention, the epidermal growth factor is hEGF.
The present invention also provides a kind of expression vectors, and nucleotide sequence and binary plant including epidermal growth factor carry
Body.
In some specific embodiments of the invention, epidermal growth factor described in the expression vector is raw for people's epidermis
The long factor.
In some specific embodiments of the invention, the construction method of the expression vector includes the following steps:
Step 1: Xbal restriction enzyme site is added in 5 ' ends of the nucleotide sequence of epidermal growth factor, at 3 ' ends
Sacl restriction enzyme site is added in end；
Step 2: clone obtains pUC57-EGF cloning vector；
Step 3: genetic fragment EGF is obtained from the resulting cloning vector of step 2 by Xbal/Sacl, it is cloned into double base
Plant vector pCam35S obtains expression vector p35S-EGF.
Specifically, the construction method of expression vector provided by the invention are as follows: by people EGF (GenBank Accession:
A00372.1 it) is designed and synthesized by Jin Sirui (Nanjing).Xbal is added in 5 ' end of EGF sequence, is added Sacl in 3 ' ends
Point, and (Figure 1A) is cloned into pUC57-EGF carrier by Jin Sirui (Nanjing).Growth factor gene segment EGF passes through Xbal/
Sacl separates (Figure 1B) from pUC57-EGF cloning vector, and is cloned into binary plant carrier pCam35S, generates respectively instantaneous
Expression vector p35S-EGF (Fig. 2).After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.
Plant expression vector isolates EGF genetic fragment by Xbal/Sacl.
By above-mentioned plant expression constructs by being worn with Multiporator (Eppendorf, Hamburg, Germany) electricity
Hole is transformed into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over to antibiotic containing kanamycin (50mg/L)
Selective LB plate on.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/L MgSO4, pH7.2) and supplement antibiotic liquid
Culture medium (50mg/L kanamycins).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.
In the present invention, EGF cDNA sequence (GenBank No. Accession: A00372.1) such as SEQ ID No.1 institute
Show；EGF amino acid sequence is as shown in SEQ ID No.2.
The present invention also provides application of the expression vector in expression epidermal growth factor.In some tools of the invention
In body embodiment, the epidermal growth factor is hEGF.
A kind of method the present invention also provides plant as host expresses growth factor converts the expression vector
Into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing protein obtains epidermal growth factor
Son；
The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean or wheat；The organ of the plant is selected from seed, leaf, rhizome
Or whole plant.
In some specific embodiments of the invention, the epidermal growth factor is hEGF.
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, the Agrobacterium is Agrobacterium tumefaciens GV3101.
Specifically, the method for mediated by agriculture bacillus vacuum infiltration are as follows: the Agrobacterium culture suspension prepared is placed in 2L and is burnt
Cup, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterial suspension
In, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visual penetration liquid
In leaf tissue.Keep 30~60s of pressure state.It opens the system quickly to release stress, penetrates into penetrating fluid in tissue
Space.The process repeats 2~3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce tissue
It gently takes out, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering from penetrating fluid.It will processing
Sample keep 4d in the dark.
After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, other than firm middle rib region, remaining
Part shows khaki region after vacuum infiltration 4 days.In order to increase the quantity for the soil Agrobacterium for immersing leaf texture, make
With scissors from the beginning 10% romaine lettuce is cut away so that romaine lettuce leaf texture infiltrates in penetrating fluid as far as possible, and discharges.With it is longer
Vacuum exposure duration compare, the method reduce leaf tissue necrosis.
In some specific embodiments of the invention, extracting and developing protein specifically:
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 (100mMKPi, pH7.8；5mMEDTA；10mM beta -mercaptoethanol) 1~2min of blender high speed homogenate.Homogenate is adjusted
It is 8.0 to pH, with filtered through gauze, filtrate is centrifuged 15min with 10,000g at 4 DEG C to remove cell fragment.Supernatant is collected,
It is mixed with ammonium sulfate (50%), and shakes be incubated for 60min on ice.It is separated again at 4 DEG C by centrifuge (10,000g)
15min.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, suspension 60min is shaken on ice, again at 4 DEG C
15min is centrifuged with 10,000g.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 protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) of sample (5uL) thermal denaturation loads are taken
Buffer (Biorad, Hercules, CA, USA) is 4~12%Bis-TrisPlus SDS- gel
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis, is then dyed with Coomassie blue G250 (Biorad)
It takes pictures again to gel afterwards.For recombinate EGF Western Blot western blot hybridization, the recombination sample of 10ul and
The standard items (Biovision) of people hEGF are respectively 10~20%Divide on Bis-Tris Plus polyacrylamide gel
From, and on its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, will be carried out respectively with the antibody (Abcam) of anti-hEGF immune anti-
It answers, dilutes the goat anti-rabbit igg (Beyotime) of 1:10000 and horseradish peroxidase (HRP) label, dilution is respectively 1:
20000, and shown using ECL plus (Amersham Biosciences), it takes pictures to display image.
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.The present invention is stirred with blender and is homogenized, and greatlys save homogenate cost and technique.Recombination
EGF separates us by SDS-PAGE and observes that estimation molecular weight is about the band (Fig. 3 A) of 5kDa in swimming lane, in stealth
Without significantly corresponding to band in control swimming lane.Purification of samples is measured based on Bradford measuring method and spectrodensitometry control group
Protein content be 0.56mg/g.In addition, Western blot analysis also detects that the band (Fig. 3 B) of about 5kDa, observe
Molecular weight of albumen it is consistent with positive controls.
Containing 10% (v/v) fetal calf serum (FBS, Gibco) from mouse embryonic fibroblasts NIH/3T3 cell line
Dulbecco improvement Eagle culture medium (DMEM, Gibco) in 37 DEG C, in 5%CO2Middle culture.By NIH/3T3 cell
100% is grown in 24 orifice plates to converge.Serum starved cells 16h scratches the surface NIH/3T3 with sterile liquid relief syringe needle, uses PBS
Washing removal cell fragment.Then the EGF and standard items purified with the dosage of 100ng/ml stimulate cell 48h.Using aobvious
Micro mirror (Nikon) is scratching region to initial wound and cell.
Pass through the bioactivity of the EGF of cell experiment research purifying.Firstly, the recombination EGF using purifying cultivates NIH/3T3
Cell uses identical business to use EGF standard as positive control.The cell Proliferation of NIH/3T3 can be by dosage by making
It is found when recombinating EGF with the purifying of 100ng/ml dosage, it is not any to check that cell growth result shows at the time point of 48h
The NIH/3T3 cell growth of processing is poor.In contrast, at using the recombination EGF of purifying or equal positive control EGF standard
The NIH/3T3 cell well-grown of reason；They stretch sprawling (Fig. 4) in scratch surface.These results indicate that passing through romaine lettuce system
The external source EGF of Transient Expression has biological activity, and romaine lettuce system may be the batch production of bioactivity recombinant pharmaceutical proteins matter
Suitable bioreactor.
The growth time of tobacco plant for the infiltration of vacuum Agrobacterium was usually 4 to 6 weeks.The present invention using romaine lettuce as
The active platform of recombinant protein production, eliminates plant growing cycle, and the time for cultivating plant early period is greatly saved.The present invention
With plant especially romaine lettuce system expression EGF, and it is successfully separated out active foreign protein under mild conditions, demonstrate,proves
Open-birth dish expression platform can be used to produce EGF.
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 (A) shows EGF cloning vector (Jin Sirui building synthesis)；
Fig. 1 (B) shows that EGF genetic fragment digestion (Xbal/SacI) is identified；
Fig. 2 shows that plant transient expression vector p35S-EGF constructs process；Utilize restriction enzyme (XbaI/SacI) double enzymes
It cuts, cuts EGF segment respectively from Fig. 1 cloning vector, connect the site XbaI/SacI into pCam35S, generate plant transient expression
Carrier p35S-EGF；
Wherein, 35S, the CaMV 35S promoter with tobacco mosaic virus (TMV) (TMV) 5 ' UTR；NPT II, for card, that is mould
The expression of the coding nptII gene of plain resistance；Nos 3 ', terminator；
Fig. 3 (A) shows the recombination fibroblastic growth using polyacrylamide gel electrophoresis (SDS-PAGE) detection purifying
The factor；Swimming lane 1: purifying recombination EGF (5 μ g)；Swimming lane 2: positive control EGF (5 μ g)；
Fig. 3 (B) shows the recombination fibroblast growth factor of Western marking hybridization check purifying；Swimming lane 1: purifying weight
Group EGF (5 μ g)；Swimming lane 2: antivacuum infiltration blade eluent negative control；
Fig. 4 shows that cell proliferation is tested；Scratch NIH/3T3 cell is handled using the EGF of purifying, carries out healing measurement；48 is small
Shi Hou, recombinant cell growth factor are obviously promoted cell-proliferation activity.
Specific embodiment
Application the invention discloses plant especially romaine lettuce as host in expression epidermal growth factor, this field skill
Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.Method of the invention and
Using being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and scope
It is interior that method described herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention is quick, transient expression recombinant protein studies have shown that romaine lettuce system can be effectively expressing platform
Provide method.Vacuum Agrobacterium permeating method is simple, quickly, reduces blade necrosis, and recombinant protein yield can be improved.
Romaine lettuce can increase protein output by bearing vacuum pressure, and every leaf is allowed more completely to permeate.Due to romaine lettuce
Be easy to grow and can commercial mass production, 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-effectiveness is higher.Present invention demonstrates that this method can be used for the short time
Interior large-scale production EGF recombinant protein.
The present invention provides plant especially romaine lettuce as host expression growth factor in application in it is raw materials used and
Reagent 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
In order to provide high efficient expression of the foreign protein in plant, the construction method of expression vector provided by the invention are as follows:
People EGF (GenBank No. Accession: A00372.1) is designed and synthesized by Jin Sirui (Nanjing).In 5 ' end of EGF sequence
Xbal is added, the site Sacl is added in 3 ' ends, and be cloned into pUC57-EGF carrier by Jin Sirui (Nanjing).Growth factor
Genetic fragment EGF is separated from pUC57-EGF cloning vector by Xbal/Sacl, and is cloned into binary plant carrier
PCam35S generates transient expression vector p35S-EGF respectively.Three kinds of plant expression constructs are passed through into use respectively
Multiporator (Eppendorf, Hamburg, Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.By institute
Bacterial strain is obtained equably to spread on the selective LB plate of antibiotic containing kanamycin (50mg/L).28 DEG C of incubations in the dark
After 2d, picking single colonie is inoculated into 0.5L YEB, and (yeast extract meat soup, 5g/L sucrose, 5g/L tryptone, 6g/L yeast are mentioned
Take object, 0.24g/LMgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation
With 25~28 DEG C of incubation 72h in oscillator (220rpm).Pass through addition YEB culture medium measure OD600 value and be adjusted to 3.5~
4.5.Then culture solution is collected, (4500 revolving speed) 10min is centrifuged.By agrobatcerium cell be resuspended in osmotic medium (10mM MES,
10mM MgSO4) in O.D.600 be 0.5.
The clone EGF genetic fragment (Fig. 1), and construct two kinds of plant expression vectors based on dual factors virus
P35S-EGF (Fig. 2).After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.
The vacuum infiltration of 2 mediated by agriculture bacillus of embodiment
Present invention optimizes the methods of Agrobacterium vacuum infiltration.The Agrobacterium culture suspension prepared is placed in 2L to burn
Cup, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterial suspension
In, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visual penetration liquid
In leaf tissue.Keep 30~60s of pressure state.It opens the system quickly to release stress, penetrates into penetrating fluid in tissue
Space.The process repeats 2~3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce tissue
It gently takes out, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering from penetrating fluid.It will processing
Sample keep 4d in the dark.
After infiltration, most romaine lettuce groups are flooded during being woven in vacuum immersion, other than firm middle rib region, remaining
Part shows khaki region after vacuum infiltration 4 days.In order to increase the quantity for the soil Agrobacterium for immersing leaf texture, make
With scissors from the beginning 10% romaine lettuce is cut away so that romaine lettuce leaf texture infiltrates in penetrating fluid as far as possible, and discharges.With it is longer
Vacuum exposure duration compare, the method reduce leaf tissue necrosis.
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；10mMercaptoethanol) 1~2min of blender high speed homogenate.By homogenate
Being adjusted to pH is 8.0, and with filtered through gauze, filtrate is centrifuged 15min 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 15min.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, suspension 60min is shaken on ice, again at 4 DEG C
Under with 10,000g be centrifuged 15min.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.
4 PAGE gel electrophoresis of embodiment and the hybridization of Western Blot western blot
The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) of sample (5uL) thermal denaturation loads are taken
Buffer (Biorad, Hercules, CA, USA) is 4~12%Bis-TrisPlus SDS- gel
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis, is then dyed with Coomassie blue G250 (Biorad)
It takes pictures again to gel afterwards.For recombination EGFWestern Blot western blot hybridization, the recombination sample of 10ul and people
HEGF standard items (Biovision) are respectively 10~20%It is separated on Bis-Tris Plus polyacrylamide gel, and
By on its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, it is immunoreacted respectively with the antibody (Abcam) of anti-hEGF, it is dilute
The goat anti-rabbit igg (Beyotime) of 1:10000 and horseradish peroxidase (HRP) label are released, dilution is respectively 1:
20000, and shown using ECL plus (Amersham Biosciences), it takes pictures to display image.
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 EGF
It is separated by SDS-PAGE.Observe that estimation molecular weight is about the band (Fig. 3 A) of 5kDa in swimming lane, in stealth control swimming
Without significantly corresponding to band in road.Albumen based on Bradford measuring method and spectrodensitometry control group measurement purification of samples
Content is respectively 0.56mg/g.In addition, Western blot analysis also detects that the about band of 5kDa (Fig. 3 B), observation
The molecular weight of albumen arrived is consistent with positive controls.
The experiment of 5 cell proliferation of embodiment
Containing 10% (v/v) fetal calf serum (FBS, Gibco) from mouse embryonic fibroblasts NIH/3T3 cell line
Dulbecco improvement Eagle culture medium (DMEM, Gibco) in 37 DEG C, in 5%CO2Middle culture.By NIH/3T3 cell
100% is grown in 24 orifice plates to converge.Serum starved cells 16h scratches the surface NIH/3T3 with sterile liquid relief syringe needle, uses PBS
Washing removal cell fragment.Then the EGF and standard items purified with the dosage of 100ng/ml stimulate cell 48h.Using aobvious
Micro mirror (Nikon) is scratching region to initial wound and cell.
Pass through the bioactivity of the haEGF of cell experiment research purifying.Firstly, the recombination EGF using purifying cultivates NIH/
3T3 cell uses identical business to use EGF standard as positive control.The cell Proliferation of NIH/3T3 can be passed through by dosage
It is found when recombinating EGF using the purifying of 100ng/ml dosage, checks that cell growth result shows not appoint at the time point of 48h
The NIH/3T3 cell growth of where reason is poor.In contrast, using the recombination EGF of purifying or equal positive control EGF standard
The NIH/3T3 cell well-grown of processing；They stretch sprawling (Fig. 4) in scratch surface.These results indicate that passing through romaine lettuce system
The external source EGF of system Transient Expression has biological activity, and romaine lettuce system may be that bioactivity recombinant pharmaceutical proteins matter batch is raw
The suitable bioreactor produced.
Embodiment 6
Control group: leaf tobacco production EGF is utilized；
Experimental group: romaine lettuce provided by the invention produces EGF；
1 hEGF of table
*Show P≤0.05 compared with the control group；#Show P≤0.01 compared with the control group；
As shown in Table 1, compared with the tobacco leaf system of control group, romaine lettuce transient expression hEGF (EGF), significantly
(P≤0.05) shortens the production cycle, and significant (P≤0.05) improves protein content, and it is living that significant (P≤0.05) improves albumen
Property, the complexity of protein purification is simplified, extremely significant (P≤0.01) 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, hEGF 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>Shenzhen Hui Sheng Biotechnology Co., Ltd
<120>application of the plant as host in expression epidermal growth factor
<130> MP1717896
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 168
<212> DNA
<213> EGF
<400> 1
atgaacagcg actctgaatg cccgctgagc catgatggct actgcctgca cgacggtgta 60
tgcatgtata tcgaagctct ggacaaatac gcatgcaact gcgtagttgg ttacatcggc 120
gaacgttgcc agtaccgcga cctgaaatgg tgggagctcc gttaataa 168
<210> 2
<211> 54
<212> PRT
<213> EGF
<400> 2
Met Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu
1 5 10 15
His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys
20 25 30
Asn Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu
35 40 45
Lys Trp Trp Glu Leu Arg
50
Claims (10)
1. application of the plant as host in expression epidermal growth factor；The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean
Or wheat；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
2. application according to claim 1, which is characterized in that the epidermal growth factor is hEGF.
3. a kind of expression vector, which is characterized in that nucleotide sequence and binary plant carrier including epidermal growth factor.
4. expression vector according to claim 3, which is characterized in that the epidermal growth factor is human epidermal growth factor
Son.
5. expression vector according to claim 3 or 4, which is characterized in that its construction method includes the following steps:
Step 1: Xbal restriction enzyme site is added in 5 ' ends of the nucleotide sequence of epidermal growth factor, adds in 3 ' ends
Enter Sacl restriction enzyme site；
Step 2: clone obtains pUC57-EGF cloning vector；
Step 3: genetic fragment EGF being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant
Carrier pCam35S obtains expression vector p35S-EGF.
6. according to application of the described in any item expression vectors of claim 3 to 5 in expression epidermal growth factor.
7. application according to claim 6, which is characterized in that the epidermal growth factor is hEGF.
8. a kind of method of plant as host expresses epidermal growth factor, which is characterized in that will be as any such as claim 3 to 5
Expression vector described in is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing egg
White matter obtains epidermal growth factor；
The plant is selected from romaine lettuce, Chinese cabbage, corn and soybean or wheat；The organ of the plant is selected from seed, leaf, rhizome or whole
Strain plant.
9. according to the method described in claim 8, it is characterized in that, the epidermal growth factor is hEGF.
10. method according to claim 8 or claim 9, which is characterized in that the mediated by agriculture bacillus vacuum infiltration includes following step
It is rapid:
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 |
|---|---|---|---|---|
| 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 |
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