CN110218249A - Plant production takes orally recombinant human granulocyte colony stimulating factor - Google Patents
Plant production takes orally recombinant human granulocyte colony stimulating factor Download PDFInfo
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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
- C07K14/52—Cytokines; Lymphokines; Interferons
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- 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
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- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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Abstract
The present invention relates to field of biotechnology, in particular to plant production takes orally recombinant human granulocyte colony stimulating factor.The present invention produces bioactive substance using plant such as romaine lettuce as the effective expression platform of recombinant protein production, using simple and efficient expression system.Capsule then is made in the blade for producing active material freeze-drying.This capsule can keep bioactivity being stored at room temperature.Hypoglycemic activity albumen successful expression is determined using Western Blot protein hybridization method.Biological activity test is the result shows that the recombinant human granulocyte colony stimulating factor activity using platform technology production is significant.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant production takes orally recombinant human granulocyte colony stimulating factor.
Background technique
Filgrastim (G-CSF) is the one kind generated by monocyte, fibroblast, endothelial cell
The glycoprotein regulatory factor that molecular weight is about 20KD, isoelectric point are about 6.0.G-CSF acts on neutrophilic granulocytes of bone marrow system
Hemopoietic forebody cell promotes its proliferation, differentiation, mature and release.In mature human body G-CSF have 177 amino acid (a) types and
174 amino acid (b) types, two kinds of isomers, the helical content of b type molecule is very high, and crystal configuration a spiral containing there are four belongs to length
Chain helical cytokine family.Biological activity and stability in view of b type are all remarkably higher than a type, and b type molecule is deep by researcher
High praise.Recombinant human g-csf is widely used in the treatment of bone-marrow transplantation and tumour, in addition, recombinant human granulocyte colony stimulation because
Son also be used to treat the diseases such as acute leukemia, alpastic anemia.Effect master of the G-CSF in terms of adjuvant therapy treatment
If increasing number of white blood cells, the risk of chemotherapy and radiation is reduced.There are many recombinant humangranulocytes based on b type at present
Preparation goes through to list, such as Filgrastim (filgrastim), Lenograstim (lenograstim), Nartograstim
(nartograstim).Wherein Filgrastim and Nartograstim are the recombined human G- of the nonglycosylated form of Bacillus coli expression
CSF, and Lenograstim is the expression of CH0 cell with glycosylation modified G-CSF.Filgrastim amino acid sequence is i.e. in natural G-
The N-terminal of CSF adds a methionine, and Nartograstim is the mutant of Filgrastim, is related to 5 amino acid in natural G-CSF altogether
Rite-directed mutagenesis.
The method for preparing GM-CSF being currently known mainly has two kinds: (1) being expressed using eukaryocyte.Such as use yeast
Cell is expressed with COS-1 zooblast, is expressed with Chinese hamster ovary celI or with insect cell.
Great advantage using eukaryotic cell expression albumen is that the albumen of expression can be glycosylated partially or completely, thus more
Close to native protein configuration.But the disadvantage is that expression quantity is generally lower, and the production cost is very high, production technology is complicated.(2) it utilizes
Escherichia coli prepare GM-CSF.Since glycosylation has no meaning to the biological activity of GM-CSF, then using Escherichia coli
(E.coli) expression of GM-CSF is just very suitable.Major part leukine is derived from E.coli at present.Escherichia coli system
The advantages of standby GM-CSF is low cost and high expression.When at present using Escherichia coli preparation GM-CSF, it is all made of endochylema representation,
Expressed GM-CSF exists in the form of insoluble occlusion body (inclusion bodies) in Escherichia coli endochylema.It is this
In terms of the shortcomings that expression system, is mainly manifested in following two: must (a) use denaturant or detergent in purification process, need
It is denaturalized the process with renaturation, this brings difficulty to the purification work in downstream, while also reducing the final yield of product；(b)
The bigger disadvantage of this expression system is to make the N-terminal of the GM-CSF of expression methionine residues more than natural GM-CSF
(Met).Body may be stimulated to generate antibody when therefore applying in vivo, to affect the treatment and increase side effect.
Therefore it provides a kind of method for easily and efficiently producing GM-CSF has important practical significance.
Summary of the invention
In view of this, the present invention provides the application that plant production takes orally recombinant human granulocyte colony stimulating factor.The present invention
The efficient platform technology produced using plant especially romaine lettuce as recombinant protein, express recombinant human granulocyte colony stimulation because
Son.And oral recombinant human granulocyte colony stimulating factor is made.The present invention ties the active peptides with blood sugar reducing function
Structure transformation and modification make its acquisition that can carry out absorbing and reaching in vivo the characteristic of effective treatment concentration by enteron aisle, and pass through
Plant produces the active material.
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's production recombinant human granulocyte colony stimulating factor；The plant choosing
From romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, root
Stem or whole plant.
On the basis of the studies above, the present invention provides a kind of expression vectors, including recombinant human granulocyte colony to stimulate
The nucleotide sequence and chloroplast expression vector of the factor.
In some specific embodiments of the invention, the sequence of the recombinant human granulocyte colony stimulating factor is that will weigh
The codon optimization of group Filgrastim is the codon of favorite plant, obtains optimization.
In some specific embodiments of the invention, the optimization is as shown in SEQ ID No.2；The recombined human
The amino acid sequence of granulocyte colony stimulating factor is as shown in SEQ ID No.1.
In some specific embodiments of the invention, construction method includes the following steps:
Step 1: being the codon of favorite plant by the codon optimization of recombinant human granulocyte colony stimulating factor, obtain excellent
Change sequence；
Step 2: the optimization that step 1 is obtained is cloned into pUC57 carrier, obtains pUC57-CSF.
The present invention also provides application of the expression vector in expression recombinant human granulocyte colony stimulating factor.
A kind of method the present invention also provides plant as host expresses recombinant human granulocyte colony stimulating factor, by institute
The expression vector stated biolistic bombardment blade obtains regeneration plant after expressing in plant chloroplast, plant leaf blade is lyophilized
It crushes, recombinant human granulocyte colony stimulating factor is made in filling capsule.
In some specific embodiments of the invention, the plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, jade
Rice, soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
In some specific embodiments of the invention, the biolistic bombardment includes the following steps:
Step 1: preparing conversion carrier
Step 2: preparing particle bullet；
Step 3: biolistic bombardment；
Step 4: being cultivated after conversion, be regenerated as plant.
The present invention is that will contain target in the way of biolistic bombardment, homologous recombination plant chloroplast expression technology
The plasmid of albumen is transferred in plant chloroplast, obtains the technology of high efficient expression in the gene plant chloroplaset.With zooblast
Expression system is compared, and the cost of plant expression system is very low, only its one thousandth to 2/1000ths.
The present invention utilizes plant leaf blade, produces oral recombinant human granulocyte colony stimulating factor.The antihypelipidemic product does not need
Injection mitigates the pain of sufferer, while this product is long-acting antihypelipidemic product, and sufferer can accomplish that medication in one week is primary.Romaine lettuce
Without containing plant noxious material, and this product is not required to protein purification process, can greatly shorten production cycle and production cost.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows carrier pUC57-CSF schematic diagram；
Fig. 2 shows western-blot result；
Fig. 3 shows that romaine lettuce expression G-CSF can significantly improve the mouse abnormal behavior of MPTP induction.
Specific embodiment
Application the invention discloses plant as host in expression recombinant human granulocyte colony stimulating 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 provides plant as host in the application for expressing recombinant human granulocyte colony stimulating factor.Preferably,
The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from
Seed, leaf, rhizome or whole plant.The present invention also provides a kind of expression vector, including recombinant human granulocyte colony stimulation because
Subsequence and carrier.
In some specific embodiments of the invention, the recombinant human granulocyte colony stimulating factor codon optimization is
The codon of favorite plant.
In some specific embodiments of the invention, the amino of the recombinant human granulocyte colony stimulating factor of the optimization
Acid sequence is as shown in SEQ ID No.1；The nucleotide sequence of the recombinant human granulocyte colony stimulating factor of the optimization such as SEQ
Shown in ID No.2.
In some specific embodiments of the invention, the carrier is plant chloroplast carrier.
In some specific embodiments of the invention, the construction method of the expression vector includes the following steps:
Step 1: being the codon of favorite plant by the codon optimization of recombinant human granulocyte colony stimulating factor；
Step 2: gene chemical synthesis being carried out by Jin Sirui and is cloned into pUC57 carrier, pUC57-CSF cloning vector is obtained；
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention pierces recombinant human granulocyte colony
Swash factor amino acid sequence and utilizes anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_
Backtranseq/ nucleotide sequence) is obtained, and is the codon of favorite plant by its codon optimization, by Jin Sirui company
(Nanjing, China) synthesis.And it is grand into pUC57 carrier by golden Stryker, it obtains pUC57-CSF carrier (Fig. 1).
The present invention also provides application of the expression vector in expression recombinant human granulocyte colony stimulating factor.
By expression vector provided by the invention biolistic bombardment plant leaf blade, plant leaf blade is harvested simultaneously after being regenerated as plant
Oral recombinant human granulocyte colony stimulating factor is made.
Plant provided by the invention is used in the application in expression recombinant human granulocyte colony stimulating factor as host
Raw material and reagent are available on the market.
Below with reference to embodiment, the present invention is further explained:
The building of 1 chloroplast expression vector of embodiment
For the high efficient expression by foreign protein in plant, by recombinant human granulocyte colony stimulating factor amino acid sequence
Nucleotide is obtained using anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/)
Sequence, and be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.
2 converting material of embodiment prepares
By vegetable seeds sterile water soaked overnight, used aseptic water washing 1 time after being impregnated 1 minute with 70% ethyl alcohol；It uses again
2%NaClO (adding 0.1%Tween-20) is handled 15 minutes, soft mixing in every 5 minutes 1 time, aseptic water washing 4-5 times；With sterile
Plantation is placed in illumination box in (containing 3% sucrose, 0.7% agar powder, pH value 5.8) on 1/2MS culture medium after filter paper blots
In 25 DEG C, 16h illumination 8h dark culturing can be used for converting for about 3 weeks.
3 particle gun of embodiment prepares
50-60mg bronze (0.6 μm) is weighed in dry 1.5mL sterilizing EP centrifuge tube.1mL dehydrated alcohol, vortex 2 is added
Minute.1mL sterile water is added, is vortexed 1 minute, is placed at room temperature for 1 minute, supernatant is removed in 10,000rpm centrifugations 2 minutes.1mL is added
Bronze is resuspended in 50% glycerol, and -20 DEG C freeze.The bronze suspension that glycerol saves state is vortexed 5 minutes and bronze is resuspended.Take 50 μ L
Bronze suspension is vortexed 1 minute in sterile 1.5mL centrifuge tube.10 μ g Plasmid DNA are added, are vortexed 30 seconds.50 μ L 2.5M are added
CaCl2 is vortexed 30 seconds.20 μ L 0.1M spermidines are added, mixture is vortexed 5 minutes, stands 2 minutes on ice.60 μ L are added to be pre-chilled
Dehydrated alcohol, finger, which flicks, to be allowed to be resuspended, and 14,000rpm centrifugations 10 seconds are removed supernatant, are repeated once.50 μ L dehydrated alcohols are added
It is resuspended, it is spare.
4 biolistic bombardment of embodiment
A certain number of carrier films are measured according to sample number, can split that film, stopping net, (note: carrier film can split film and need every rifle
Replacement, stopping to net same sample can share) it is impregnated 15 minutes in dehydrated alcohol, with aseptic water washing 2 times, naturally dry,
It is spare.The carrier film dried is put into sterile iron hoop, is flattened.The bullet prepared vortex is mixed well, 10 μ L bullets are taken
In carrier film center, naturally dry.Corpuscular emission device is removed bombardment room, lid is screwed off, is added and stops net, particle slide glass
It is mounted in fixing groove (fine-grained one down), screws on lid, corpuscular emission device is put back into bombardment room.
Embodiment 5 is cultivated after converting and screening
1. dark culture: the romaine lettuce blade after bombardment being cut, the leaf dish for being cut into 10~20mm2 is placed in RMOL culture medium (no
Added with antibiotic) in 25 DEG C dark culture 2 days.
2. screening and culturing: the material that dark culture is terminated is transferred in screening and culturing medium (antibiotic concentration is 50 μ g/mL)
Carry out screening and culturing.
3. culture of rootage: bud is transferred to root induction in root media (antibiotic concentration is 100 μ g/mL).
6 Western blot testing goal protein expression situation of embodiment
Vegetable protein is extracted using liquid nitrogen grinding, denatured lysis, cracking 5 × sample-loading buffer of supernatant (is added using preceding
Enter beta -mercaptoethanol to it is final concentration of 5%) in 4:1 ratio mixing (such as 200 μ l protein cleavage supernatants and 50 μ 5 × loadings of l delay
Fliud flushing mixing), it mixes, 95 DEG C of heating 6min, while handling negative control and positive control；Electrophoretic voltage spacer gel 80V, separation
Glue 120V is run after destination protein to separation gel middle position, stops electrophoresis, is recycled lower slot electrophoresis liquid, is dismantled electrophoretic apparatus, press
According to cathode (black), sponge, filter paper, gel, pvdf membrane (1 × transfer is soaked in after being washed in advance with methanol activation 15s, ddH2O
In buffer) or NC film (being not required to activate), filter paper, sponge, anode (transparent) sequence place, exhaust bubble after assemble, be put into electricity
Swimming slot (note black corresponds to electrophoresis tank black and is put on one side), fills it up with transfering buffering liquid, entire electrophoresis tank is put into ice water mixed liquor
In, 90V electrophoresis 1.0h；5% skimmed milk power (confining liquid) is prepared at the end of electrophoresis is fast, and the film after transfer is put into room in confining liquid
Temperature closing at least 1h, 4 DEG C of incubation primary antibodies are overnight (primary antibody is diluted in 5% skimmed milk power, thinner ratio reference book)；It uses
PBST or TBST washs 15min × 3 time, and incubation at room temperature 1~2h of secondary antibody, PBST or TBST are washed 15min × 3 time, tried using DAB
Agent box develops the color, and takes pictures, and analyzes destination protein expression: as shown in Figure 1, non-express romaine lettuce does not have destination protein detection,
Expression plant can detect destination protein band, and size meets expection, it was demonstrated that CSF is expressed in romaine lettuce blade.
7 recombinant human granulocyte colony stimulating factor Activity determination of embodiment
1- methyl 4-phenyl -1,2,3,6- tetrahydropyridines (MPTP), can specificity damage dopaminergic neuron,
There is the symptom of similar Parkinson's disease (PD).C57/BL6J mouse is chosen in experiment, and male, 20-22g, 12-14 weeks, animal was raised
It supports in 24 ± 2 DEG C of room temperature of environment, maintains the dark 12 hours cycle alternations of light-.30 mouse are randomly divided into 3 groups, and every group 10,
Respectively solvent control group, MPTP model group, MPTP+ romaine lettuce express CSF group.MPTP is injected intraperitoneally with the dosage of 30mg/kg, even
Continuous injection 5 days, CSF are that 40ug/kg oral administration is 5 days continuous (from the 7th day).Solvent control group is physiological saline.12nd day
Behavioral assessment is carried out to mouse.
Coarse bar top (diameter 8mm, high 55cm) is placed on by mouse is soft upwards.From the beginning mouse adjusts upward to complete
The downward time is recorded as T1, and mouse is recorded as T2 from the time for moving downwardly to four limbs and all arriving at bar bottom.Every mouse weight
Reinspection is surveyed 5 times and is averaged.
Table 1 statistics indicate that the result shows that, romaine lettuce expression G-CSF can significantly improve MPTP induction mouse abnormal behavior.Such as
Fig. 3.
1 CSF of table improves MPTP effect duration
*Show P≤0.05 compared with the control.
8 animal toxicity test of embodiment
The experimental white mouse of 7 weeks sizes is randomly divided into three treatment groups, every group 10, receives contain hypoglycemic egg respectively
White (according to weight feeding 500ng/g) (recombinant human granulocyte colony stimulating factor that the present invention obtains), and be free of blood sugar reducing proteins
One of two kinds of experiment capsules, receive identical experimental diet.Continuous feeding 10 days is observed after each feeding, often in fact
It needs to be observed continuously 6 hours or more, does not see that mouse is in excitatory state or holddown, is not slow in action
Phenomena such as, also there is not situations such as diarrhea.Prove that recombinant human granulocyte colony stimulating factor oral administration safety is high.
Embodiment 9
Experimental group 1: plant production recombinant human granulocyte colony stimulating factor provided by the invention；
Control group 1: recombinant human granulocyte colony stimulating factor is produced using yeast cells；
Experimental group 2: leaf tobacco production recombinant human granulocyte colony stimulating factor is utilized；
2 recombinant human granulocyte colony stimulating factor of table
*Show P≤0.05 compared with control group 1；**Show P≤0.01 compared with control group 1；
#Show P≤0.05 compared with experimental group 2；##Show P≤0.01 compared with experimental group 2；
As shown in Table 2, experimental group 1 is compared with the animal system of control group 1, romaine lettuce transient expression recombination provided by the invention
Filgrastim, simplifies the complexity of protein purification, and extremely significant (P≤0.01) reduces production cost.
Experimental group 1 is compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression recombinant human granulocyte colony stimulating factor,
The complexity of protein purification is simplified, significant (P≤0.05) reduces production cost.
Compared with the control group, tobacco leaf transient expression recombinant human granulocyte colony stimulating factor is than animal system letter for experimental group 2
The complexity of protein purification is changed, significant (P≤0.05) reduces production cost.
In summary test result show botanical system especially romaine lettuce system be it is more economical, efficiently express platform.
Can quick transient expression recombinant protein, recombinant human granulocyte colony stimulating factor 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>Wang Yueju
<120>plant production takes orally recombinant human granulocyte colony stimulating factor
<130> MP1909370
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 207
<212> PRT
<213> Recombinant human granulocyte colony-stimulating factor
<400> 1
Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln
1 5 10 15
Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro
20 25 30
Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu
35 40 45
Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys
50 55 60
Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu
65 70 75 80
Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser
85 90 95
Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His
100 105 110
Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile
115 120 125
Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala
130 135 140
Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala
145 150 155 160
Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala
165 170 175
Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser
180 185 190
Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro
195 200 205
<210> 2
<211> 624
<212> DNA
<213> Recombinant human granulocyte colony-stimulating factor
<400> 2
atggccggcc ccgccaccca gagccccatg aagctgatgg ccctgcagct gctgctgtgg 60
cacagcgccc tgtggaccgt gcaggaggcc acccccctgg gccccgccag cagcctgccc 120
cagagcttcc tgctgaagtg cctggagcag gtgaggaaga tccagggcga cggcgccgcc 180
ctgcaggaga agctggtgag cgagtgcgcc acctacaagc tgtgccaccc cgaggagctg 240
gtgctgctgg gccacagcct gggcatcccc tgggcccccc tgagcagctg ccccagccag 300
gccctgcagc tggccggctg cctgagccag ctgcacagcg gcctgttcct gtaccagggc 360
ctgctgcagg ccctggaggg catcagcccc gagctgggcc ccaccctgga caccctgcag 420
ctggacgtgg ccgacttcgc caccaccatc tggcagcaga tggaggagct gggcatggcc 480
cccgccctgc agcccaccca gggcgccatg cccgccttcg ccagcgcctt ccagaggagg 540
gccggcggcg tgctggtggc cagccacctg cagagcttcc tggaggtgag ctacagggtg 600
ctgaggcacc tggcccagcc ctga 624
Claims (9)
1. the application that plant produces recombinant human granulocyte colony stimulating factor as host；The plant be selected from romaine lettuce, spinach, kind
Eggplant, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
2. a kind of expression vector, which is characterized in that nucleotide sequence and leaf including recombinant human granulocyte colony stimulating factor
Green body expression vector.
3. expression vector according to claim 2, which is characterized in that the sequence of the recombinant human granulocyte colony stimulating factor
It is classified as the codon by the codon optimization of recombinant human granulocyte colony stimulating factor for favorite plant, obtains optimization.
4. expression vector according to claim 3, which is characterized in that the optimization is as shown in SEQID No.2；
The amino acid sequence of the recombinant human granulocyte colony stimulating factor is as shown in SEQ ID No.1.
5. according to the described in any item expression vectors of claim 2 to 4, which is characterized in that its construction method includes the following steps:
Step 1: being the codon of favorite plant by the codon optimization of recombinant human granulocyte colony stimulating factor, obtain optimization sequence
Column；
Step 2: the optimization that step 1 is obtained is cloned into pUC57 carrier, obtains pUC57-CSF.
6. according to the described in any item expression vectors of claim 2 to 6 in expression recombinant human granulocyte colony stimulating factor
Using.
7. a kind of method of plant as host expresses recombinant human granulocyte colony stimulating factor, which is characterized in that will be such as right
It is required that 2 to 5 described in any item expression vectors biolistic bombardment blades, obtain regeneration after expressing in plant chloroplast and plant
Strain, plant leaf blade is lyophilized and is crushed, and recombinant human granulocyte colony stimulating factor is made in filling capsule.
8. the method for claim 7, which is characterized in that the plant be selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage,
Corn and soybean, wheat or tobacco；The organ of the plant is selected from seed, leaf, rhizome or whole plant.
9. according to the method described in claim 8, it is characterized in that, the biolistic bombardment includes the following steps:
Step 1: preparing conversion carrier
Step 2: preparing particle bullet；
Step 3: biolistic bombardment；
Step 4: being cultivated after conversion, be regenerated as plant.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1251772A (en) * | 1999-09-27 | 2000-05-03 | 上海健神生物工程有限公司 | Process for extracting granulocyte stimulating factor and its compound polyose capsules |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1251772A (en) * | 1999-09-27 | 2000-05-03 | 上海健神生物工程有限公司 | Process for extracting granulocyte stimulating factor and its compound polyose capsules |
Non-Patent Citations (2)
| Title |
|---|
| MEHDI SHARIFI TABAR等: "Human Granulocyte Colony-Stimulating Factor (hG-CSF) Expression in Plastids of Lactuca sativa", 《IRANIAN BIOMEDICAL JOURNAL》 * |
| 无: "NCBI Reference Sequence: NP_000750.1", 《GENEBANK》 * |
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Application publication date: 20190910 |