CN107254486A

CN107254486A - Application of the romaine lettuce as host in expression growth factor

Info

Publication number: CN107254486A
Application number: CN201710458332.8A
Authority: CN
Inventors: 王跃驹; 李文; 焦顺昌
Original assignee: Shenzhen Rise Biotechnology Co Ltd
Current assignee: Beijing Ruicheng Haiway Health Technology Co. Ltd.
Priority date: 2017-06-16
Filing date: 2017-06-16
Publication date: 2017-10-17

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

Application of the romaine lettuce as host in expression growth factor

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 disease^TM(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 GeneArt^TMGeneOptimizer^TM(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 GeneArt^TMGeneOptimizer^TM(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.

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 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

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

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<170> PatentIn version 3.3

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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

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；

Repeat the above steps 2~3 times, lucifuge processing 4d.