CN110204620A - Application of the plant as host in expression MGLP fusion protein - Google Patents
Application of the plant as host in expression MGLP fusion protein Download PDFInfo
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- CN110204620A CN110204620A CN201910549759.8A CN201910549759A CN110204620A CN 110204620 A CN110204620 A CN 110204620A CN 201910549759 A CN201910549759 A CN 201910549759A CN 110204620 A CN110204620 A CN 110204620A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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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
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Abstract
The present invention relates to field of biotechnology, in particular to plant is as host in the application for expressing MGLP fusion protein.The present invention expresses MGLP fusion protein using simple and effective mediated by agriculture bacillus vacuum infiltration methods using plant such as romaine lettuce as the effective expression platform of recombinant protein production.The expression system determines that plant foreign protein can be collected after Agrobacterium is infected 4 days.MGLP fusion protein successful expression is determined using Western Blot protein hybridization method, goes out MGLP fusion protein with AKTA protein purification system successful purification.Biological activity test is the result shows that significantly reduce the blood sugar concentration in dog blood using the MGLP fusion protein that the platform technology produces.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant is expressing answering in MGLP fusion protein as host
With.
Background technique
Diabetes are common disease, the frequently-occurring disease characterized by chronic hyperglycemia, are lacked by internal insulin secretion or effect
Fall into, or both exist simultaneously caused by sugar, fat, protein metabolism disorder.Clinically mainly there is insulin-dependent
(IDDM, I type) and non-insulin-depending type (NIDDM, II type) two types.With the improvement of living standards, no matter in flourishing state
Still in developing country, the disease incidence of diabetes is all being risen year by year for family.Diabetes are as a kind of serious non-infectious slow
Property disease become one of the great public health problem of whole world various countries close attention, be in global range after cardiovascular and
Third killer after tumor disease.From the point of view of the data that the World Health Organization announces, nineteen ninety-five whole world diabetic is only
30000000 people or so, and 1.35 hundred million were had increased to by 1997, will there are 300,000,000 type 2 diabetes patients, patient's amplification to the year two thousand thirty
Most fast area is Asia and Africa.The conventional treatment model of type 2 diabetes patient is usually to follow diet control, oral anti-
The escalation therapy of broncho of diabetes medicament and exogenous insulin.But there are still many to be resolved in current treating diabetes field
Major issue, but also there are some side effects and limitations.
Glucagon-like-peptide-1 (Glucagon-like peptide-1, GLP-1) is secreted by Endocrine Cells In The Gut
Duodenin, be Proglucagon gene translation post-processing product, in vivo there are many existence form.It has following
Physiological action: acting on beta Cell of islet with glucose-dependent manner, promotes the transcription of insulin gene, increases the life of insulin
Object synthesis and secretion；The proliferation and differentiation of β cell are stimulated, β Apoptosis is inhibited, to increase beta Cell of islet quantity, inhibits pancreas
The secretion of glucagons, appetite-suppressing and ingests, and delays gastric content emptying etc..These functions all advantageously reduce postprandial blood sugar
And blood glucose is made to maintain constant level.
Although natural GLP-1 has many advantages, such as that its Half-life in vivo is only 2 minutes or so in treatment diabetes,
Limit its direct application clinically.
Summary of the invention
In view of this, the application the present invention provides plant as host in expression MGLP fusion protein.Benefit of the invention
The efficient platform technology for using plant especially romaine lettuce to produce as recombinant protein, expresses MGLP fusion protein.And mild
Under conditions of be successfully separated out active foreign protein, it was demonstrated that plant especially romaine lettuce expression platform can successfully be used to produce
MGLP fusion protein.Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, the potential disease pest of infection human body is eliminated
Evil etc..Production cost is substantially reduced, Product Safety is improved.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides MGLP fusion proteins, include
(I), the amino acid sequence as shown in SEQ ID No.1；Or
(II), the amino acid sequence as described in (I) is substituted, lacks or adds the amino that one or more amino acid obtain
Acid sequence, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (I)；Or
(III), with the amino acid sequence of (I) or (II) described sequence at least 80% homology.
On the basis of the studies above, the present invention provides the nucleotide for encoding the MGLP fusion protein, have
(I), the nucleotide sequence as shown in SEQ ID No.2；Or
(II), the complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID 2；Or
(III), with the nucleotide sequence coded same protein of (I) or (II), but due to the degeneracy of genetic code with
(I) or the different nucleotide sequence of the nucleotide sequence of (II)；Or
(IV), one or more nucleosides are substituted, lack or added with nucleotide sequence shown in (I), (II) or (III)
Acid sequence obtain nucleotide sequence, and with nucleotide sequence nucleosides functionally identical or similar shown in (I), (II) or (III)
Acid sequence；Or
(V), with the nucleotide sequence of (I), (II), (III) or (IV) described nucleotide sequence at least 80% homology.
In addition, the present invention also provides expression vector, including the nucleotide and carrier to be transformed.
In some specific embodiments of the invention, the carrier to be transformed is chloroplast expression vector.
The present invention also provides the construction methods of the expression vector, include the following steps:
Step 1: being respectively the codon of favorite plant by the codon optimization of the MGLP fusion protein, obtain optimization
The sequence of MGLP fusion protein；Its nucleotide sequence is as shown in SEQ ID No.2；
Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the MGLP fusion protein of the optimization,
Sac I site is added in 3 ' ends；
It is cloned into pUC57 carrier, obtains pMGLP cloning vector；
Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant
Carrier pCam35S obtains expression vector p35S-MGLP.
Plant transient expression technology is will to contain mesh using a variety of different technical approach in plant growth to certain phase
The plasmid of mark albumen is transferred in plant cell, and efficient, controllable expression system is established in plant cell, it is short to obtain the gene
The technology of temporary controlled expression.It is short the time required to transient expression compared with stablizing expression, it does not need exogenous origin gene integrator to place
In main plant chromosome, it is only necessary to which several days time can take experimental result.Compared with bacterial expression system, plant expression system
Expressed albumen can correctly be folded, process, modify, protein active produced is higher than bacterial expression system；
Compared with animal cell expression system, the cost of plant expression system is very low, only its one thousandth to 2/1000ths.
The present invention also provides the expression vector or plant in expression MGLP fusion protein or preparation comprising described
Application in the drug of MGLP fusion protein；The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, small
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 drug is oral hypoglycemic agents.
The present invention also provides host, conversion has the plant or microorganism of the expression vector；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 strain
Plant.
The present invention also provides drugs, including the fusion protein and pharmaceutically acceptable auxiliary material.
In some specific embodiments of the invention, the drug is oral hypoglycemic agents.
A kind of method the present invention also provides plant as host expresses MGLP fusion protein, by the expression vector
It is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing protein obtains MGLP and melts
Hop protein.
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 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.
It can will extend partly declining for it under the conditions of guaranteeing that its is active after certain amino acid mutations in natural GLP-1
Phase, normal blood glucose level can be kept by accomplishing to be administered once a week.The Related product listed at present has
Liraglutide, Dulaglutide, Semaglutide etc..The present invention is by transferrins and GLP-1 amalgamation and expression, Ke Yishi
Present long-acting hypoglycemic mitigates sufferer long term frequent injection bring pain.
The present invention is using plant such as romaine lettuce as the effective expression platform of recombinant protein production, using simple and have
The mediated by agriculture bacillus vacuum infiltration methods of effect express MGLP fusion protein.The expression system determines plant foreign protein in agriculture bar
Bacterium can collect after infecting 4 days.MGLP fusion protein successful expression is determined using Western Blot protein hybridization method, is used
AKTA protein purification system successful purification goes out MGLP fusion protein.Biological activity test using the platform technology the result shows that produced
MGLP fusion protein significantly reduce the blood sugar concentration in dog blood.
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 cloning vector pMGLP schematic diagram；
Fig. 2 shows that MGLP fusion protein plant binary expression vector p35S-MGLP constructs process；Utilize restriction enzyme
(Xbal/SacI) double digestion cuts MGLP fusion protein from Fig. 1 cloning vector, connects the site Xbal/SacI into pCam35S,
Generate plant binary expression vector p35S-MGLP；
LB and RB:Ti plasmid right boundary；35S, the CaMV 35S with tobacco mosaic virus (TMV) (TMV) 5 ' UTR start
Son；NPT II, the expression of the coding nptII gene for kalamycin resistance；Nos 3 ', terminator；
Fig. 3 shows PAGE gel electrophoresis result；Swimming lane 1: non-to infect romaine lettuce；Swimming lane 2: the MGLP of romaine lettuce expression merges egg
It is white；Swimming lane 3:MGLP fusion protein commercially produced product.
Specific embodiment
A kind of application the invention discloses plant as host in expression MGLP fusion protein, those skilled in the art
Present disclosure can be used for reference, realization of process parameters is suitably modified.In particular, it should be pointed out that all similar substitutions and modifications pair
It is it will be apparent that they are considered as being included in the present invention for those skilled in the art.Method and application of the invention is
Through being described by preferred embodiment, related personnel obviously can not depart from the content of present invention, in spirit and scope to this
Methods and applications described in text are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
In view of this, the application the present invention provides plant as host in expression MGLP fusion protein.Benefit of the invention
The efficient platform technology for using plant especially romaine lettuce to produce as recombinant protein, expresses MGLP fusion protein.And mild
Under conditions of be successfully separated out active foreign protein, it was demonstrated that plant especially romaine lettuce expression platform can successfully be used to produce
MGLP fusion protein.Time is short (4d), and purifying is simple, and it is convenient to produce.Gene contamination is eliminated, the potential disease pest of infection human body is eliminated
Evil etc..Production cost is substantially reduced, Product Safety is improved.
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 MGLP fusion protein.Preferably, 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.The present invention also provides a kind of expression vectors, sequence and carrier including MGLP fusion protein.
In some specific embodiments of the invention, the sequence of the MGLP fusion protein is by MGLP fusion protein
Codon optimization is the codon of favorite plant, the MGLP fusion protein sequence of the optimization of acquisition.
In some specific embodiments of the invention, the nucleotide sequence such as SEQ of the MGLP fusion protein of the optimization
Shown in ID No.1；The amino acid sequence of the MGLP fusion protein of the optimization is as shown in SEQID No.2.
In some specific embodiments of the invention, the carrier is binary plant 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 MGLP fusion protein, obtain the MGLP fusion of optimization
The sequence of albumen；
Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the MGLP fusion protein of the optimization,
Sac I site is added in 3 ' ends；
It is grand into pUC57 carrier by golden Stryker, obtain pMGLP cloning vector；
Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant
Carrier pCam35S obtains expression vector p35S-MGLP.
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by people's MGLP fusion protein amino
Acid sequence is obtained using anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/)
Nucleotide sequence, and be the codon of favorite plant by its codon optimization, by Jin Sirui company (Nanjing, China) synthesis.?
Xbal restriction enzyme site is added in the 5 ' end of MGLP fusion protein sequence of optimization, and the site Sacl is added in 3 ' ends.And by
Golden Stryker is grand into pUC57 carrier, obtains pMGLP cloning vector (Fig. 1).Genetic fragment is carried by XbaI/Sacl from clone
It is separated in body, and is cloned into binary plant carrier pCam35S, generated plant expression vector p35S-MGLP (Fig. 2).
The present invention also provides application of the expression vector in expression MGLP fusion protein.
In addition, a kind of method the present invention also provides plant as host expresses MGLP fusion protein, the present invention is mentioned
The expression vector of confession is transformed into Agrobacterium, after entering plant tissue by mediated by agriculture bacillus vacuum infiltration, extracting and developing albumen
Matter obtains MGLP fusion protein.
Specifically, by plant expression vector p35S-MGLP by with Multiporator (Eppendorf, Hamburg,
Germany) Electroporation Transformation is into Agrobacterium tumefaciens GV3101.Obtained strains are equably spread over containing kanamycin anti-
On the selective LB plate of raw element (50mg/L).In the dark after 28 DEG C of incubation 2d, picking single colonie is inoculated into 0.5L YEB (ferment
Female 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).The culture of inoculation is incubated in oscillator (220rpm) with 25~28 DEG C
Educate 72h.OD600 value is measured by addition YEB culture medium and is adjusted to 3.5~4.5.Then culture solution is collected, is centrifuged (4500 turns
Speed) 10min.It is 0.5 that agrobatcerium cell, which is resuspended in osmotic medium (10mM MES, 10mM MgSO4) to O.D.600,.
It will prepare that mix containing p35S-MGLP Agrobacterium equivalent to O.D.600 be 0.5；Culture suspension is placed in
2L beaker, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterium and is hanged
In supernatant liquid, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visible infiltration
Transparent liquid is in leaf tissue.Keep 30~60s of pressure state.It opens the system quickly to release stress, makes penetrating fluid infiltration group
Knit interior space.The process repeats 2~3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce
Tissue gently takes out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering.It will
The sample of processing keeps 4d in the dark.
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, Agrobacterium is specially Agrobacterium tumefaciens GV3101.
Clone's pMGLP genetic fragment of the present invention, and binary plant expression vector p35S-MGLP (Fig. 2) is constructed,
After completing construct, being digested with specific restriction enzyme confirms that genetic fragment is complete.After infiltration, most romaine lettuce groups are woven in
It is flooded during vacuum immersion, other than firm middle rib region, rest part shows after vacuum infiltration 4 days khaki
Region.
Extracting and developing protein specifically: the romaine lettuce sample through Agrobacterium vacuum infiltration is stirred with blender, and uses body
Product is than Extraction buffer (100mM KPi, the pH7.8 for 1:1 ratio；5mM EDTA；10mM beta -mercaptoethanol) it is high in blender
1~2min of speed homogenate.Homogenate is adjusted to pH8.0, with filtered through gauze, filtrate 4 DEG C with 10,000g be centrifuged 15min with
Remove cell fragment.Supernatant is collected, is mixed with ammonium sulfate (50%), and shakes be incubated for 60min on ice.Pass through centrifuge
(10,000g) separates 15min at 4 DEG C again.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, is shaken on ice
Dynamic suspension 60min, is centrifuged 15min at 4 DEG C again with 10,000g.Then, liquid is discarded supernatant, sample pellet albumen will be handled
Matter is dissolved in 5mL buffer (20mM KPi, pH 7.8；2mM EDTA；10m M beta -mercaptoethanol) in and store at 4 DEG C.
PAGE gel electrophoresis specifically: collect the protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce, sampling
(95 DEG C) load buffers (Biorad, Hercules, CA, USA) of product (5 μ L) thermal denaturation are in 4-12%Bis-Tris
Plus SDS- denaturant gel (ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, non denatured
The affine degree of antibody is detected in gel electrophoresis.Then gel is clapped again after being dyed with Coomassie blue G250 (Biorad)
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.We are stirred with blender and are homogenized, and greatly save homogenate cost and technique.Recombinate MGLP
Fusion protein separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about 92kDa (Fig. 3) in swimming lane
Band, meet MGLP fusion protein molecular weight of albumen.It is pure based on Bradford measuring method and the measurement of spectrodensitometry control group
The protein content for changing sample is about 1.78mg/g.
The present invention is using romaine lettuce come transient expression MGLP fusion protein, and (4d) can produce high-content in a relatively short period of time
Protein.Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this
Kind method reduces bio-safety problem to the maximum extent, because processed romaine lettuce tissue is usually in completely enclosed facility
Or developed in container, biological pollution problem is not present.Romaine lettuce does not contain plant noxious material, and itself protein content is few,
Conducive to the protein purification in downstream.Using romaine lettuce system production MGLP fusion protein, the production cycle can be greatly shortened and be produced into
This.
The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, be
A kind of method of quick transient expression recombinant protein.The vacuum Agrobacterium permeating method that the present invention describes is simple, quickly, and
And recombinant protein yield can be improved.Romaine lettuce can increase protein output by bearing vacuum pressure, and allow every leaf
The more complete infiltration of son.Due to romaine lettuce be easy to grow and can commercial mass production, than other transient expression plants, such as
Tobacco etc. is easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can be significantly reduced.It is comprehensive
Upper described, the present invention, which can use, is mass produced MGLP fusion protein in the romaine lettuce system short time.
The raw materials used and reagent in the application in expression MGLP fusion protein is equal as host for plant provided by the invention
It is available on the market.
Below with reference to embodiment, the present invention is further explained:
The building of 1 plant transient expression vector of embodiment
For the high efficient expression by foreign protein in plant, by MGLP fusion protein amino acid sequence using it is counter translate it is soft
Part (https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) obtains nucleotide sequence, and its is close
Numeral is optimized for the codon of favorite plant, by Jin Sirui company (Nanjing, China) synthesis.In the MGLP fusion protein sequence of optimization
It arranges the end 5' and Xbal restriction enzyme site is added, the site Sacl is added in the end 3'.And pUC57 is cloned by Jin Sirui company
It in carrier, obtains pMGLP cloning vector (Fig. 1), genetic fragment is separated from cloning vector by Xbal/Sacl, and is cloned into
Binary plant carrier, pCam35S are generated plant expression vector p35S-MGLP (Fig. 2).Plant expression vector is passed through into use
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 vacuum infiltration of 2 mediated by agriculture bacillus of embodiment
It will prepare that mix containing p35S-MGLP Agrobacterium equivalent to O.D.600 be 0.5.Culture suspension is placed in
2L beaker, is placed in drier.The romaine lettuce that this laboratory saves is inverted (core is upward) and lightly rotates on bacterium and is hanged
In supernatant liquid, drier is sealed.Vacuum pump (Welch Vacuum, Niles, IL, USA) is opened to evacuate, and visible infiltration
Transparent liquid is in leaf tissue.It is kept for pressure state 30~60 seconds.It opens the system quickly to release stress, makes penetrating fluid infiltration group
Knit interior space.The process repeats 2 to 3 times, until high-visible penetrating fluid is spread obviously in romaine lettuce tissue.Then by romaine lettuce
Tissue gently takes out from penetrating fluid, and three times with distilled water continuous flushing, is then transferred into the container of plastic foil covering.It will
The sample of processing is kept 4 days in the dark.
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；10m M beta -mercaptoethanol) homogenate of blender high speed 1-2 minutes.By homogenate
It is adjusted to pH 8.0, with filtered through gauze, filtrate is centrifuged 15 minutes 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 15 minutes.Obtained supernatant is subjected to the second wheel ammonium sulfate (70%) precipitating, shakes suspend 60 minutes on ice, again 4
With 10,000g centrifugation 15 minutes at DEG C.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 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.
4 PAGE gel electrophoresis of embodiment
The protein purification extracted from Agrobacterium vacuum infiltration romaine lettuce is collected, (95 DEG C) of thermal denaturation of sample (5 μ L) loads are taken
Buffer (Biorad, Hercules, CA, USA) is 4~12%Bis-TrisPlus SDS- denaturant gel
(ThermoFisher Scientific, Waltham, MA, USA) runs electrophoresis.Equally, it is detected in native gel electrophoresis anti-
The affine degree of body.Then it takes pictures again to gel after being dyed with Coomassie blue G250 (Biorad).Recombinate MGLP fusion
Albumen separates us by denaturant gel SDS-PAGE and observes that estimation molecular weight is about the item of 92kDa (Fig. 3) in swimming lane
Band meets MGLP fusion protein molecular weight of albumen.Purified sample is measured based on Bradford measuring method and spectrodensitometry control group
The protein content of product is about 1.81mg/g.
The protein active of 5 MGLP fusion protein of embodiment detects
After continuing seven weeks stationary phases, dog is randomly divided into Liang Ge treatment group, every group 3, reception contains blood sugar reducing proteins
(MGLP fusion protein made from embodiment 4) and without one of two kinds of experiment capsules of blood sugar reducing proteins, does and repeat for the first time.Again
It is secondary to be grouped dog at random, receive another different experimental diet, does second of repetition.It is at least for 2 weeks to repeat I and II,
Blood glucose response is detected after repeating every time.
Before blood sugar test starts, dog fasting 24 hours.Shave off the hair of catheter insertion site, aseptic process, conduit insertion
Right cephalic vein.Spacing of about 5 minutes two baseline samples of acquisition.After acquiring the last one baseline sample, fed immediately to dog suitable
The diet of its weight 1% simultaneously contains 1 or 3 Jiangtang capsules, it is at most allowed to eat 15 minutes.If dog does not eat in 15 minutes
Experimental diet, then the same day does not detect its blood glucose response, and next day is detected again.10,20,30,45,60,120,180 and after feed
240 minutes, acquire additional blood sample.1300 × g of blood sample is centrifuged 15 minutes, when after acquisition will be each in two hours
Between two aliquot samples of point 1ml blood plasma freeze.Plasma glucose concentration (mg/dl) is measured using hexokinase method.
The experimental result of sugared concentration in 1 dog blood of table
Note:*Show compared with no hypoglycemic control group with significant difference (P < 0.05)；**Show compared with no hypoglycemic control group and has
There is extremely significant difference (P < 0.01).
The present invention is using romaine lettuce come transient expression antibody, and (4d) can produce the protein of high-content in a relatively short period of time.
Romaine lettuce is higher plant, can carry out posttranslational modification process, that is, the albumen expressed is automatically active.And this method is most
Reduce to limits bio-safety problem, because processed romaine lettuce tissue is usually in completely enclosed facility or container
Biological pollution problem is not present in exploitation.Romaine lettuce does not contain plant noxious material, and itself protein content is few, is conducive to downstream
Protein purification.Using romaine lettuce system production, production cycle and production cost can be greatly shortened
6 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) (the MGLP fusion protein that the present invention obtains), commercially (is pressed with Suo Malutai Semaglutid
According to weight feeding 500ng/g) it is used as positive control, and without one of two kinds of experiment capsules of blood sugar reducing proteins, receive identical
Experimental diet.Continuous feeding 10 days is observed after each feeding in fact, needs to be observed continuously daily 6 hours or more, not see
Mouse is in excitatory state or holddown and situations such as diarrhea does not also occur phenomena such as not being slow in action.It proves
The fusion protein capsule oral of Suo Malutai Semaglutide and Lumbrokinase is highly-safe.
Embodiment 7
Control group: MGLP fusion protein is produced using zooblast；
Experimental group 1: plant production MGLP fusion protein provided by the invention；
Experimental group 2: leaf tobacco production MGLP fusion protein is utilized；
2 MGLP fusion protein of table
*Show P≤0.05 compared with the control group；**Show P≤0.01 compared with the control group；
#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, romaine lettuce transient expression MGLP provided by the invention
Fusion protein, extremely significant (P≤0.01) shorten the production cycle, and extremely significant (P≤0.01) improves protein content, simplifies egg
The complexity of white purifying, extremely significant (P≤0.01) reduce production cost.
Experimental group 1 is compared with the tobacco leaf system of experimental group 2, romaine lettuce transient expression MGLP fusion protein, significant (P≤0.05)
The production cycle is shortened, significant (P≤0.05) improves protein content, simplifies the complexity of protein purification, extremely significant (P
≤ 0.01) production cost is reduced.
Compared with the control group, tobacco leaf transient expression MGLP fusion protein (P≤0.05) more significant than animal system contracts experimental group 2
The short production cycle, the complexity of protein purification is simplified, significant (P≤0.05) reduces production cost.In summary it tries
Test the result shows that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform.It being capable of quick transient expression weight
MGLP fusion protein can be mass produced in histone matter 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>application of the plant as host in expression MGLP fusion protein
<130> MP1906701
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2523
<212> DNA
<213>MGLP fusion protein (MGLP fusion protein)
<400> 1
atgggcaagc agatggctgc tctttgcggt ttccttcttg tggctctttt gtggcttacc 60
cctgatgtgg ctcatgctca tggtgaggga acctttacct ccgacgtgtc atcttacctt 120
gaaggccaag ctgcccaaga gttcattgct tggcttgtgg atggtagaca cggcgaggga 180
acttttacta gcgacgtgag ttcttacctc gagggtcaag cagctcaaga attcatagcc 240
tggttggttg atggaaggca tggcgaaggc acctttacca gtgatgtgtc ctcttacttg 300
gaaggtcagg ccgctcaaga gtttatcgca tggctcgttg atggacgtca cggtgagggc 360
acttttacct ctgatgtgag cagttacctg gaaggacagg cagcacaaga attcattgca 420
tggctggtag acggaagagg tggtggtgga tctggtggcg gaggttctgg cggtggtggt 480
tctgttcctg ataagactgt taggtggtgc gctgtttcag agcatgaggc tactaagtgc 540
cagagcttca gggaccatat gaagtctgtg atccctagcg acggtccttc tgttgcttgt 600
gtgaagaagg ctagctacct ggattgcatc agggctattg ctgctaacga ggctgatgct 660
gtgactcttg atgctggtct tgtgtacgat gcttacctgg ctcctaacaa ccttaagcct 720
gttgtggctg agttctacgg cagcaaagaa gatcctcaga ccttctacta cgctgtggct 780
gtggttaaga aggacagcgg ctttcagatg aaccagctga ggggtaagaa gtcttgccat 840
actggtcttg gtaggtccgc tggttggaat atccctattg gtctgctgta ctgcgatctg 900
cctgaaccta gaaagcctct tgagaaggct gtggccaact tcttctctgg atcttgtgct 960
ccttgcgctg atggcactga ttttccacag ctttgtcagc tttgccctgg ttgcggttgc 1020
tctactctta accagtactt cggttacagc ggcgctttca agtgccttaa ggatggtgct 1080
ggtgatgtgg cattcgtgaa gcactctacc atcttcgaga acctggctaa caaggccgat 1140
agggatcagt acgagcttct gtgccttgac aacaccagaa agcctgtgga tgagtacaag 1200
gattgccacc ttgctcaggt gccatctcat actgtggtgg ctagatccat gggtggcaaa 1260
gaggatctta tctgggagct tctgaaccag gctcaagagc acttcggcaa ggacaagtct 1320
aaagagttcc agctgttcag cagccctcac ggtaaggatc tgctgttcaa ggattctgct 1380
cacggcttcc ttaaggtgcc acctagaatg gacgctaaga tgtacctggg ctacgagtac 1440
gttaccgcca ttaggaatct tagagagggg acttgtccag aggctcctac tgatgaatgc 1500
aagccagtta agtggtgtgc cttgtctcat cacgagaggc tgaagtgtga tgagtggtct 1560
gtgaacagcg tgggcaagat tgagtgtgtg tctgctgaaa ctaccgagga ctgcattgcc 1620
aagatcatga acggtgaggc tgacgctatg tctctggatg gtggattcgt gtacattgct 1680
ggtaagtgcg gtcttgtgcc tgtgcttgct gagaactacg agaagtctga taactgcgag 1740
gatacccctg aggctggtta ctttgctgtt gcagtggtga agaagtccgc ttctgatctg 1800
acctgggata acctgaaggg caagaagtca tgtcacaccg ctgttggtag aactgctggc 1860
tggaatatcc caatgggcct cctgtacaac aagatcaacc actgcaggtt cgacgagttc 1920
ttctcagaag gttgcgctcc tggcagcaag aaagatagct ctttgtgcaa gctgtgcatg 1980
ggctctggtc ttaatctttg cgagccgaac aacaaagagg gctactacgg ttacactggg 2040
gcttttagat gcctggtcga gaagggtgat gtcgcctttg ttaagcacca gactgtgcct 2100
cagaataccg gtggtaagaa tcctgatcct tgggccaaga acctgaacga gaaggattac 2160
gagctgcttt gcctggatgg tactcgtaag ccagttgaag agtacgctaa ctgccatctt 2220
gctagggctc ctaatcatgc tgtggtgacc agaaaggaca aagaggcttg cgtccacaag 2280
attcttaggc agcagcagca cctgttcggt tctgaagtta ctgattgcag cggcaacttc 2340
tgcctgttca ggtctgagac taaggacctg cttttcaggg acgataccgt gtgcttggct 2400
aagcttcatg accggaacac ctatgagaag tacctcggtg aggaatacgt gaaggctgtt 2460
ggcaatctga ggaagtgcag cacctcttca cttttggagg cttgcacttt ccgtcggcct 2520
tga 2523
<210> 2
<211> 840
<212> PRT
<213>MGLP fusion protein (MGLP fusion protein)
<400> 2
Met Gly Lys Gln Met Ala Ala Leu Cys Gly Phe Leu Leu Val Ala Leu
1 5 10 15
Leu Trp Leu Thr Pro Asp Val Ala His Ala His Gly Glu Gly Thr Phe
20 25 30
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe
35 40 45
Ile Ala Trp Leu Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser
50 55 60
Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala
65 70 75 80
Trp Leu Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser Asp Val
85 90 95
Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu
100 105 110
Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
115 120 125
Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asp
130 135 140
Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu
165 170 175
Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro
180 185 190
Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp
195 200 205
Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp
210 215 220
Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro
225 230 235 240
Val Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr
245 250 255
Tyr Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln
260 265 270
Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly
275 280 285
Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg
290 295 300
Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala
305 310 315 320
Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro
325 330 335
Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala
340 345 350
Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His
355 360 365
Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr
370 375 380
Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys
385 390 395 400
Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser
405 410 415
Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln
420 425 430
Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser
435 440 445
Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu
450 455 460
Lys Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr
465 470 475 480
Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro
485 490 495
Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu
500 505 510
Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu
515 520 525
Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn
530 535 540
Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala
545 550 555 560
Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Glu Lys Ser
565 570 575
Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val
580 585 590
Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys
595 600 605
Lys Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro
610 615 620
Met Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe
625 630 635 640
Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys
645 650 655
Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys
660 665 670
Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys
675 680 685
Gly Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly
690 695 700
Gly Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr
705 710 715 720
Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala
725 730 735
Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys
740 745 750
Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu
755 760 765
Phe Gly Ser Glu Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg
770 775 780
Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala
785 790 795 800
Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr
805 810 815
Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu
820 825 830
Glu Ala Cys Thr Phe Arg Arg Pro
835 840
Claims (10)
1.MGLP fusion protein, which is characterized in that it is included
(I), the amino acid sequence as shown in SEQ ID No.1；Or
(II), the amino acid sequence as described in (I) is substituted, lacks or adds the amino acid sequence that one or more amino acid obtain
Column, and with the amino acid sequence functionally identical or similar of amino acid sequence shown in (I)；Or
(III), with the amino acid sequence of (I) or (II) described sequence at least 80% homology.
2. encoding the nucleotide of MGLP fusion protein as described in claim 1, which is characterized in that have
(I), the nucleotide sequence as shown in SEQ ID No.2；Or
(II), the complementary nucleotide sequence of the nucleotide sequence as shown in SEQ ID 2；Or
(III), with the nucleotide sequence coded same protein of (I) or (II), but due to the degeneracy of genetic code with (I) or
(II) the different nucleotide sequence of nucleotide sequence；Or
(IV), one or more nucleotides sequences are substituted, lack or added with nucleotide sequence shown in (I), (II) or (III)
Arrange obtain nucleotide sequence, and with nucleotide sequence nucleotides sequence functionally identical or similar shown in (I), (II) or (III)
Column；Or
(V), with the nucleotide sequence of (I), (II), (III) or (IV) described nucleotide sequence at least 80% homology.
3. expression vector, which is characterized in that including nucleotide as claimed in claim 2 and carrier to be transformed.
4. expression vector as claimed in claim 3, which is characterized in that the carrier to be transformed is chloroplast expression vector.
5. the construction method of expression vector as described in claim 3 or 4, which comprises the steps of:
Step 1: being respectively the codon of favorite plant by the codon optimization of the MGLP fusion protein, obtain the MGLP of optimization
The sequence of fusion protein；Its nucleotide sequence is as shown in SEQ ID No.2；
Step 2: Xbal restriction enzyme site is added in 5 ' ends of the sequence of the MGLP fusion protein of the optimization, at 3 ' ends
Sac I site is added in end；
It is cloned into pUC57 carrier, obtains pMGLP cloning vector；
Step 3: genetic fragment being obtained from the resulting cloning vector of step 2 by Xbal/Sacl, is cloned into binary plant carrier
PCam35S obtains expression vector p35S-MGLP.
6. expression vector or plant as described in claim 3 or 4 in expression MGLP fusion protein or is prepared comprising the MGLP
Application in the drug of fusion protein；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.
7. application as claimed in claim 6, which is characterized in that the drug is oral hypoglycemic agents.
8. host, which is characterized in that conversion has the plant or microorganism of the expression vector as described in claim 3 or 4；The plant
Selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant be selected from seed, leaf,
Rhizome or whole plant.
9. drug, which is characterized in that including fusion protein as described in claim 1 and pharmaceutically acceptable auxiliary material.
10. drug as claimed in claim 9, which is characterized in that the drug is oral hypoglycemic agents.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020259111A1 (en) * | 2019-06-24 | 2020-12-30 | 王跃驹 | Application of fabricating oral hypoglycemic capsule from fusion protein of transferrin and plant-produced glucagon-like peptide-1 oligopeptide |
-
2019
- 2019-06-24 CN CN201910549759.8A patent/CN110204620B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| CALMETTES,C.等: "3VE1_B", 《GENBANK》 * |
| FANGFANG XU等: "Bioactivity of a modified human Glucagon-like peptide-1", 《PLOS ONE》 * |
| MARTIN E. BRANDSMA等: "Plant-derived recombinant human serum transferring demonstrates multiple functions", 《PLANT BIOTECHNOLOGY JOURNAL》 * |
| TRACEY A. RUHLMAN: "Plastid Transformation in Lettuce ( Lactuca sativa L.) by Biolistic DNA Delivery", 《METHODS IN MOLECULAR BIOLOGY》 * |
| 于源华: "《生物工程与技术专业基础实验教程》", 30 June 2016, 北京理工大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020259111A1 (en) * | 2019-06-24 | 2020-12-30 | 王跃驹 | Application of fabricating oral hypoglycemic capsule from fusion protein of transferrin and plant-produced glucagon-like peptide-1 oligopeptide |
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