CN109456399A - Application of the plant as host in expression keratinocyte growth factor - Google Patents
Application of the plant as host in expression keratinocyte growth factor Download PDFInfo
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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 keratinocyte growth factor.The present invention expresses human horny cell growth factor-2 (human keratinocyte growth factor, abbreviation KGF) 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 KGF successful expression is determined using SDS-PAGE method, Western blot (Western method).Cell proliferation is experiments have shown that the KGF of 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 KGF of mass production.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant especially romaine lettuce as host answering in expressing K GF
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).
Keratinocyte growth factor (KGF), also referred to as FGF7 appear in the healing phases of wound epithelial cell formation.
In this stage, keratinocyte covers wound, forms epithelial cell.Keratinocyte growth factor molecular weight is 18.9kDa, is contained
163 amino acid.Its 39% sequence is similar to Basic Fibroblast Growth Factor.Its table in some stromal fibroblast cells systems
It reaches, but is not expressed in normal spongiocyte and epithelial cell line.KGF stimulation and keratinocyte and epithelial cell etc.
The differentiation and proliferation of cell.KGF Porcine HGF can also promote hair healthy to grow.
Currently, KGF is produced in Escherichia coli.Expression and production system of the plant as pharmaceutical protein, have been 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 humans and animals disease
Substance travels to the risk of the mankind during generate protein.In addition, plant eukaryotic albumen inner membrance expression system and secretion
Approach is similar to mammalian cell.Plant expression system can mass production macromolecule and multi-subunit pharmaceutical protein,
And it is significantly better than prokaryotic expression system, such as escherichia expression system.As needed posttranslational modification, glycosylated egg
Monoclonal antibody that is white, and needing to assemble, cannot be realized with prokaryotic system.Using the pharmaceutical protein of plant production as life
Object reagent has been commercialized, or the vaccine additive as poultry.2012, food and drug administration (FDA) approval
For treating the albumen ELELYSO of 1 type Gaucher disease of genetic diseaseTM(taliglucerase alfa), and this albumen is benefit
It is produced with carrot.In the past decade, demand of the people to pharmaceutical protein sharply increases, so FDA approval is for clinic
The quantity of the plant medical protein of 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, are grown using romaine lettuce as host in expression
The factor has important practical significance.
Summary of the invention
In view of this, the present invention, which provides plant especially romaine lettuce as host, is expressing answering in keratinocyte growth factor
With.The active platform that the present invention is produced using romaine lettuce as recombinant protein, eliminates plant growing cycle, and early period is greatly saved
Cultivate the time of plant.Present invention romaine lettuce system expression KGF and to be successfully separated out active under mild conditions
Foreign protein, it was demonstrated that romaine lettuce expression platform can be used to produce KGF.
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 keratinocyte growth factor；The plant is selected from life
Dish, 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 keratinocyte growth factor is human Keratiocyte growth factor 1
Son.
The present invention also provides a kind of expression vectors, nucleotide sequence and binary plant including keratinocyte growth factor
Carrier.
In some specific embodiments of the invention, the keratinocyte growth factor is human Keratiocyte growth factor 1
Son.
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 keratinocyte growth factor,
Sacl restriction enzyme site is added in 3 ' ends；
Step 2: clone obtains pUC57-KGF cloning vector；
Step 3: genetic fragment KGF being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into double base
Plant vector pCam35S obtains expression vector p35S-KGF.
Specifically, the construction method of expression vector provided by the invention are as follows: by KGF (GenBank Accession:
AAA67335.1 it) is designed and synthesized by Jin Sirui (Nanjing).Xbal is added in 5 ' end of KGF sequence, Sacl is added in 3 ' ends
Site, and (Figure 1A) is cloned into pUC57-KGF carrier by Jin Sirui (Nanjing).Growth factor gene segment KGF passes through Xbal/
Sacl separates (Figure 1B) from pUC57-KGF cloning vector, and is cloned into binary plant carrier pCam35S, generates respectively instantaneous
Expression vector p35S-KGF (Fig. 2).After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.
Plant expression vector isolates KGF genetic fragment by Xbal/Sacl.
By plant expression constructs by being turned with Multiporator (Eppendorf, Hamburg, Germany) electroporation
Change into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over to the choosing of antibiotic containing kanamycin (50mg/L)
On selecting property LB plate.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.By agriculture
Bacilli-cell is resuspended in osmotic medium (10mM MES, 10mM MgSO4) in O.D.600 be 0.5.
In the present invention, KGF cDNA sequence (GenBank No. Accession: AAA67335.1) such as SEQ ID No.1
It is shown；KGF amino acid sequence is as shown in SEQ ID No.2.
The present invention also provides application of the expression vector in expression keratinocyte growth factor.Of the invention one
In a little specific embodiments, the keratinocyte growth factor is human horny cell growth factor-2.
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 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.
In some specific embodiments of the invention, keratinocyte growth factor described in the method is that people's cutin is thin
The intracellular growth factor.
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 (100mM KPi, pH7.8；5mM EDTA；10mM beta -mercaptoethanol) 1~2min of blender high speed homogenate.By homogenate tune
Section is 8.0 to pH, 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 60min 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.
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-Tris Plus 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 KGF Western Blot western blot hybridization, the recombination sample of 10ul and
The standard items (Biovision) of people hKGF are respectively 10~20%Divide on Bis-Tris Plus polyacrylamide gel
From, and by its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, with anti-hEGF, the antibody (Abcam) of hKGF and hIGF
It is immunoreacted respectively, dilutes the goat anti-rabbit igg (Beyotime) of 1:10000 and horseradish peroxidase (HRP) label,
Dilution is respectively 1:20000, and is shown using ECL plus (Amersham Biosciences), is clapped display image
According to.
The Downstream processing of the recombinant protein of plant origin is typically difficult to and valuableness, because cellulose cell wall is difficult to split
Solution and secondary plant metabolites.The present invention is stirred with blender and is homogenized, and greatlys save homogenate cost and technique.Recombination
KGF separates us by SDS-PAGE and observes that estimation molecular weight is about the band (Fig. 3 A) of 19kDa 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 0.59mg/g.In addition, Western blot analysis also detects that the band (Fig. 3 B) of about 19kDa, 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 KGF 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 KGF of cell experiment research purifying.Firstly, the recombination KGF using purifying cultivates NIH/3T3
Cell uses identical business to use KGF standard as positive control.The cell Proliferation of NIH/3T3 can be by dosage by making
It is found when recombinating KGF 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 KGF of purifying or equal positive control KGF 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 KGF 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 romaine lettuce system expression KGF, and it is successfully separated out active foreign protein under mild conditions, it was demonstrated that romaine lettuce expression
Platform can be used to produce KGF.
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 KGF cloning vector (Jin Sirui building synthesis)；
Fig. 1 (B) shows that KGF genetic fragment digestion (Xbal/SacI) is identified；
Fig. 2 shows that plant transient expression vector p35S-KGF constructs process；Utilize restriction enzyme (XbaI/SacI) double enzymes
It cuts, cuts KGF segment respectively from Fig. 1 cloning vector, connect the site XbaI/SacI into pCam35S, generate plant transient expression
Carrier p35S-KGF；
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 KGF (5 μ g)；Swimming lane 2: positive control KGF (5 μ g)；
Fig. 3 (B) shows the recombination fibroblast growth factor of Western marking hybridization check purifying；Swimming lane 1: purifying weight
Group KGF (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 KGF 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 romaine lettuce as host in expression growth factor, those skilled in the art can use for reference
Present disclosure is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field skill
It is it will be apparent that they are considered as being included in the present invention for art personnel.Method of the invention and application by compared with
Good embodiment is described, related personnel obviously can not depart from the content of present invention, in spirit and scope to as described herein
Methods and applications are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention is 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 KGF recombinant protein.
The institute in the present invention provides plant especially romaine lettuce as host in expression keratinocyte growth factor
It is available on the market with raw material and reagent.
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 KGF (GenBank No. Accession: AAA67335.1) is designed and synthesized by Jin Sirui (Nanjing).At 5 ' end of KGF sequence
Xbal is added in end, the site Sacl is added in 3 ' ends, and be cloned into pUC57-KGF carrier by Jin Sirui (Nanjing).Growth because
Mrna exon fragment KGF is separated from pUC57-KGF cloning vector by Xbal/Sacl, and is cloned into binary plant carrier
PCam35S generates transient expression vector p35S-KGF 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/L MgSO4, pH7.2) and supplement antibiotic liquid culture medium (50mg/L kanamycins).By the culture of inoculation
Object is in oscillator (220rpm) with 25~28 DEG C of incubation 72h.OD600 value is measured by addition YEB culture medium and is adjusted to 3.5
~4.5.Then culture solution is collected, (4500 revolving speed) 10min is centrifuged.Agrobatcerium cell is resuspended in osmotic medium (10mM
MES, 10mM MgSO4) in O.D.600 be 0.5.
The clone KGF genetic fragment (Fig. 1), and construct two kinds of plant expression vectors based on dual factors virus
P35S-KGF (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-Tris Plus 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 KGF Western Blot western blot hybridization, the recombination sample of 10ul and
People hKGF standard items (Biovision) are respectively 10~20%It is separated on Bis-Tris Plus polyacrylamide gel,
And by its electrophoretic transfer to polyvinylidene fluoride (PVDF) film, it is immunoreacted respectively with the antibody (Abcam) of anti-hKGF,
The goat anti-rabbit igg (Beyotime) of 1:10000 and horseradish peroxidase (HRP) label are diluted, 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 KGF
It is separated by SDS-PAGE.Observe that estimation molecular weight is about the band (Fig. 3 A) of 19kDa 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 0.56,0.59,0.57mg/g.In addition, Western blot analysis also detects that the about band of 19kDa
(Fig. 3 B), the molecular weight of albumen observed 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 KGF 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 haKGF of cell experiment research purifying.Firstly, the recombination KGF using purifying cultivates NIH/
3T3 cell uses identical business to use KGF standard as positive control.The cell Proliferation of NIH/3T3 can be passed through by dosage
It is found when recombinating KGF 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 KGF of purifying or equal positive control KGF 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 KGF 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 KGF is utilized；
Experimental group: romaine lettuce provided by the invention produces KGF；
1 human horny cell growth factor-2 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 keratinocyte growth factor (KGF) is shown
It writes (P≤0.05) and shortens the production cycle, significant (P≤0.05) improves protein content, and significant (P≤0.05) improves albumen
Activity, simplifies the complexity of protein purification, and 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, human horny cell growth factor-2 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 keratinocyte growth factor
<130> MP1718283
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 585
<212> DNA
<213> KGF
<400> 1
atgcacaaat ggatactgac atggatcctg ccaactttgc tctacagatc atgctttcac 60
attatctgtc tagtgggtac tatatcttta gcttgcaatg acatgactcc agagcaaatg 120
gctacaaatg tgaactgttc cagccctgag cgacacacaa gaagttatga ttacatggaa 180
ggaggggata taagagtgag aagactcttc tgtcgaacac agtggtacct gaggatcgat 240
aaaagaggca aagtaaaagg gacccaagag atgaagaata attacaatat catggaaatc 300
aggacagtgg cagttggaat tgtggcaatc aaaggggtgg aaagtgaatt ctatcttgca 360
atgaacaagg aaggaaaact ctatgcaaag aaagaatgca atgaagattg taacttcaaa 420
gaactaattc tggaaaacca ttacaacaca tatgcatcag ctaaatggac acacaacgga 480
ggggaaatgt ttgttgcctt aaatcaaaag gggattcctg taagaggaaa aaaaacgaag 540
aaagaacaaa aaacagccca ctttcttcct atggcaataa cttaa 585
<210> 2
<211> 194
<212> PRT
<213> KGF
<400> 2
Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg
1 5 10 15
Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys
20 25 30
Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser
35 40 45
Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile
50 55 60
Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp
65 70 75 80
Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn
85 90 95
Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly
100 105 110
Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys Leu Tyr
115 120 125
Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu Ile Leu
130 135 140
Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly
145 150 155 160
Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly Ile Pro Val Arg Gly
165 170 175
Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro Met Ala
180 185 190
Ile Thr
Claims (10)
1. application of the plant as host in expression keratinocyte growth factor；The plant be selected from romaine lettuce, Chinese cabbage, corn,
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 keratinocyte growth factor is human Keratiocyte growth
The factor.
3. a kind of expression vector, which is characterized in that nucleotide sequence and binary plant carrier including keratinocyte growth factor.
4. expression vector according to claim 3, which is characterized in that the keratinocyte growth factor is human keratinized cell
Growth factor.
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 keratinocyte growth factor, at 3 ' ends
Sacl restriction enzyme site is added in end；
Step 2: clone obtains pUC57-KGF cloning vector；
Step 3: genetic fragment KGF being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant
Carrier pCam35S obtains expression vector p35S-KGF.
6. according to application of the described in any item expression vectors of claim 3 to 5 in expression keratinocyte growth factor.
7. application according to claim 6, which is characterized in that the keratinocyte growth factor is human Keratiocyte growth
The factor.
8. a kind of method of plant as host expresses keratinocyte growth factor, which is characterized in that will be such as claim 3 to 5
Described in any item expression vectors are transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, are extracted, are divided
From protein, keratinocyte growth factor is obtained；
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 keratinocyte growth factor is human Keratiocyte growth
The factor.
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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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766107A (en) * | 2005-07-22 | 2006-05-03 | 成都芝田生物工程有限公司 | Recombinant human keratinized cell growth factor production method |
| CN103468737A (en) * | 2013-08-04 | 2013-12-25 | 吉林农业大学 | Plant oil body containing keratinocyte growth factor KGF2 |
| CN107083399A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in expression dissolved blood protein |
| CN107083398A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in the expression antibody of PD 1 and/or PD L1 antibody |
-
2017
- 2017-09-06 CN CN201710795718.8A patent/CN109456399A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1766107A (en) * | 2005-07-22 | 2006-05-03 | 成都芝田生物工程有限公司 | Recombinant human keratinized cell growth factor production method |
| CN103468737A (en) * | 2013-08-04 | 2013-12-25 | 吉林农业大学 | Plant oil body containing keratinocyte growth factor KGF2 |
| CN107083399A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in expression dissolved blood protein |
| CN107083398A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in the expression antibody of PD 1 and/or PD L1 antibody |
Non-Patent Citations (1)
| Title |
|---|
| LAWRENCE D.JOH等: "Evaluating Extraction and Storage of a Recombinant Protein Produced in Agroinfiltrated Lettuce", 《BIOTECHNOL.PROG.》 * |
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