CN110218259A - The application of the fusion protein of plant production glucagon-like-peptide-1 small peptide and transferrins manufacture orally-taken blood sugar reducing capsule - Google Patents
The application of the fusion protein of plant production glucagon-like-peptide-1 small peptide and transferrins manufacture orally-taken blood sugar reducing capsule Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, the in particular to application of the fusion protein of plant production glucagon-like-peptide-1 small peptide and transferrins manufacture orally-taken blood sugar reducing capsule.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 significantly reduce the blood sugar concentration in dog blood using the Jiangtang capsule that the platform technology produces.
Description
Technical field
The present invention relates to field of biotechnology, in particular to plant production glucagon-like-peptide-1 small peptide and transferrins
Fusion protein manufacture orally-taken blood sugar reducing capsule application.
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.And it will can guarantee its work after certain amino acid mutations in natural GLP-1
Extend its half-life period under conditions of property, normal blood glucose level can be kept by accomplishing to be administered once a week.At present on
The Related product in city has Liraglutide, Dulaglutide, Semaglutide etc..Due to the property of polypeptide drug itself
And the various barriers that human body generates it, conventional administration by way of always with injection based on.The present invention by transferrins with
GLP-1 amalgamation and expression may be implemented be administered orally, and mitigate sufferer long term frequent injection bring pain.
Summary of the invention
In view of this, the present invention a kind of plant production glucagon-like-peptide-1 small peptide and transferrins be provided merge egg
The application of white manufacture orally-taken blood sugar reducing capsule.The present invention carries out structure of modification and modification to the active peptides with blood sugar reducing function, makes
It obtains the characteristic that can carry out absorbing and reach in vivo effective treatment concentration by enteron aisle, and the activity is produced by plant
Substance.The efficient platform technology that the present invention is produced using plant especially romaine lettuce as recombinant protein, expresses glucagon
The fusion protein of sample peptide -1 (GLP-1) small peptide and transferrins.And orally-taken blood sugar reducing capsule is made.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides the fusion proteins of glucagon-like-peptide-1 (GLP-1) small peptide and transferrins, 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.
The present invention provides the nucleotide of encoding said 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.
On above-mentioned Research foundation, the present invention also provides expression vector, including the nucleotide and to be transformed
Carrier.
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: respectively by the password of the glucagon-like-peptide-1 (GLP-1) small peptide and the fusion protein of transferrins
Son is optimized for the codon of favorite plant, and nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pGLP-1 is obtained.
The present invention also provides the expression vector or plant expression glucagon-like-peptide-1 (GLP-1) small peptide with
The fusion protein of transferrins or preparation include the application in the drug of the 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.
In some specific embodiments of the invention, the drug is the oral preparation of hypoglycemic.
In addition, conversion has the plant or microorganism of the expression vector the present invention also provides host；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 drugs, including the fusion protein and pharmaceutically acceptable auxiliary material.
In some specific embodiments of the invention, the drug is the oral preparation of hypoglycemic.
The present invention also provides a kind of plants as host expresses glucagon-like-peptide-1 (GLP-1) small peptide and to turn iron egg
The method of white fusion protein obtains expression vector biolistic bombardment blade after expressing in plant chloroplast
Plant leaf blade freeze-drying is crushed, is extracted, obtains glucagon-like-peptide-1 (GLP-1) small peptide and transferrins by regeneration plant
Fusion protein.
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 also provides a kind of plants prepared as host hypoglycemic drug method, the expression vector is used
Biolistic bombardment blade obtains regeneration plant after expressing in plant chloroplast, and plant leaf blade freeze-drying is crushed, is extracted, is obtained
The fusion protein of glucagon-like-peptide-1 (GLP-1) small peptide and transferrins, it is filling.
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 utilizes plant leaf blade, produces orally-taken blood sugar reducing capsule.The antihypelipidemic product does not need to inject, and mitigates the pain of sufferer
Hardship, while this product is long-acting antihypelipidemic product, sufferer can accomplish that medication in one week is primary.Romaine lettuce does not contain plant Toxic
Matter, and this product is not required to protein purification process, can greatly shorten production cycle and production cost.
The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, leaf
Green body can efficient expression activity albumen.Due to romaine lettuce be easy to grow and can commercial mass production, than other plants
Object, such as tobacco are easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can significantly drop
It is low.In conclusion the present invention can use romaine lettuce system large-scale production glucagon-like-peptide-1 (GLP-1) small peptide and turn iron
The fusion protein of albumen.
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 pGLP-1 schematic diagram；
Fig. 2 shows western-blot result.
Specific embodiment
The invention discloses a kind of manufactures of the fusion protein of plant production glucagon-like-peptide-1 small peptide and transferrins
The application of orally-taken blood sugar reducing capsule, those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.Especially need
It is noted that all similar substitutions and modifications are apparent to those skilled in the art, they are considered as
It is included in the present invention.Method of the invention and application are described by preferred embodiment, and related personnel obviously can be
Do not depart from the content of present invention, in spirit and scope to method described herein and application is modified or appropriate changes and combinations,
Carry out implementation and application the technology of the present invention.
The present invention provides the applications of plant production orally-taken blood sugar reducing capsule.The present invention is using plant especially romaine lettuce as weight
The efficient platform technology of histone production, express glucagon-like-peptide-1 (GLP-1) small peptide and transferrins merges egg
It is white.And orally-taken blood sugar reducing capsule is made.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides plant as host in expression glucagon-like-peptide-1 (GLP-1) small peptide and transferrins
The application of fusion protein.Preferably, the plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or cigarette
Grass；The organ of the plant is selected from seed, leaf, rhizome or whole plant.The present invention also provides a kind of expression vectors, including pancreas
The fusion protein sequence and carrier of (GLP-1) small peptide of glucagon-like peptide -1 and transferrins.
In some specific embodiments of the invention, glucagon-like-peptide-1 (GLP-1) small peptide and turn iron egg
White fusion protein codon optimization is the codon of favorite plant.
In some specific embodiments of the invention, glucagon-like-peptide-1 (GLP-1) small peptide of the optimization with
The fusion protein sequence of transferrins is as shown in SEQ ID No.1；Glucagon-like-peptide-1 (GLP-1) small peptide of the optimization
Nucleotide sequence with the fusion protein of transferrins is as shown in SEQ 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: the codon optimization by glucagon-like-peptide-1 (GLP-1) small peptide and the fusion protein of transferrins is
The codon of favorite plant；
Step 2: gene chemical synthesis being carried out by Jin Sirui and is cloned into pUC57 carrier, pGLP-1 cloning vector is obtained；
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by glucagon-like-peptide-1
(GLP-1) the fusion protein amino acid sequence of small peptide and transferrins using anti-translation software (https: //
Www.ebi.ac.uk/Tools/st/emboss_backtranseq/ nucleotide sequence) is obtained, and is by its codon optimization
The codon of favorite plant, by Jin Sirui company (Nanjing, China) synthesis.And it is grand into pUC57 carrier by golden Stryker, it obtains
PGLP-1 carrier (Fig. 1).
The present invention also provides the expression vectors in expression glucagon-like-peptide-1 (GLP-1) small peptide and to turn iron egg
Application in white fusion protein.
By expression vector provided by the invention biolistic bombardment plant leaf blade, plant leaf blade is harvested simultaneously after being regenerated as plant
Orally-taken blood sugar reducing capsule is made.
Plant chloroplast expression technology be by biolistic bombardment, homologous recombination in the way of by the plasmid containing target protein
It is transferred in plant chloroplast, obtains the technology of high efficient expression in the gene plant chloroplaset.With animal cell expression system phase
Than the cost of plant expression system is very low, only its one thousandth to 2/1000ths.
The present invention utilizes plant leaf blade, produces orally-taken blood sugar reducing capsule.The antihypelipidemic product does not need to inject, and mitigates the pain of sufferer
Hardship, while this product is long-acting antihypelipidemic product, sufferer can accomplish that medication in one week is primary.Romaine lettuce does not contain plant Toxic
Matter, and this product is not required to protein purification process, can greatly shorten production cycle and production cost.
The present invention is found through experiments that, botanical system especially romaine lettuce system be it is more economical, efficiently express platform, leaf
Green body can efficient expression activity albumen.Due to romaine lettuce be easy to grow and can commercial mass production, than other plants
Object, such as tobacco are easier to obtain and cheaper, and due to not needing complicated special producing equipment, cost can significantly drop
It is low.In conclusion the present invention can use romaine lettuce system large-scale production glucagon-like-peptide-1 (GLP-1) small peptide and turn iron
The fusion protein of albumen.
The fusion protein manufacture of plant production glucagon-like-peptide-1 small peptide and transferrins provided by the invention is oral
Raw materials used and reagent is available on the market in the application of Jiangtang capsule.
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 glucagon-like-peptide-1 (GLP-1) small peptide and turn iron
The fusion protein amino acid sequence of albumen 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.
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 suspensions in sterile 1.5mL from
Heart pipe is vortexed 1 minute.10 μ g Plasmid DNA are added, are vortexed 30 seconds.50 μ L 2.5M CaCl2 are added, are vortexed 30 seconds.20 μ L are added
0.1M spermidine, mixture are vortexed 5 minutes, stand 2 minutes on ice.The dehydrated alcohol for adding 60 μ L to be pre-chilled, finger, which flicks, is allowed to weight
Outstanding, 14,000rpm centrifugations 10 seconds are removed supernatant, are repeated once.The resuspension of 50 μ L dehydrated alcohols is added, 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).
Embodiment 6Western blot testing goal protein expression situation
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 l5 × loading 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 2: the result shows that chloroplast transformation plant has GLP-
1 band, non transformed plants do not have band of expression, it was demonstrated that GLP-1 is expressed in romaine lettuce blade.
The fusion protein Activity determination of 7 glucagon-like-peptide-1 of embodiment (GLP-1) small peptide and transferrins
After continuing seven weeks stationary phases, dog is randomly divided into Liang Ge treatment group, every group 3, receives contain hypoglycemic respectively
Albumen (fusion protein of glucagon-like-peptide-1 made from embodiment 5 (GLP-1) small peptide and transferrins) and be free of hypoglycemic
One of two kinds of experiment capsules of albumen do and repeat for the first time.Dog is grouped at random again, receives another different experiment drink
Food, is cooked second of repetition.It is at least for 2 weeks to repeat I and II, detects blood glucose response after each repeat.
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: * shows with significant difference (P < 0.05)；* shows with extremely significant difference (P < 0.01).
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) (glucagon-like-peptide-1 (GLP-1) small peptide that the present invention obtains and transferrins melt
Hop protein), and without one of two kinds of experiment capsules of blood sugar reducing proteins, receive identical experimental diet.Continuous feeding 10 days, often
It is observed in fact after secondary feeding, needs to be observed continuously daily 6 hours or more, do not see that mouse is in excitatory state and still inhibits
Also there is not situations such as diarrhea phenomena such as not being slow in action in state.It proves glucagon-like-peptide-1 (GLP-1)
The fusion protein oral administration safety of small peptide and transferrins is high.
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, it is short that glucagon-like-peptide-1 (GLP-1) can be mass produced in a short time
The fusion protein of peptide and transferrins.
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>fusion protein of plant production glucagon-like-peptide-1 small peptide and transferrins manufactures orally-taken blood sugar reducing capsule
Using
<130> MP1908154
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 815
<212> PRT
<213>glucagon-like-peptide-1 (the fusion protein F usion protein of of GLP-1 small peptide and transferrins
glucagon-like peptide-1 GLP-1 short peptide and transferrin)
<400> 1
Met His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu
1 5 10 15
Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asp Gly Arg His
20 25 30
Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln
35 40 45
Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asp Gly Arg His Gly Glu
50 55 60
Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala
65 70 75 80
Gln Glu Phe Ile Ala Trp Leu Val Asp Gly Arg His Gly Glu Gly Thr
85 90 95
Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu
100 105 110
Phe Ile Ala Trp Leu Val Asp Gly Arg Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Val Pro Asp Lys Thr Val Arg Trp
130 135 140
Cys Ala Val Ser Glu His Glu Ala Thr Lys Cys Gln Ser Phe Arg Asp
145 150 155 160
His Met Lys Ser Val Ile Pro Ser Asp Gly Pro Ser Val Ala Cys Val
165 170 175
Lys Lys Ala Ser Tyr Leu Asp Cys Ile Arg Ala Ile Ala Ala Asn Glu
180 185 190
Ala Asp Ala Val Thr Leu Asp Ala Gly Leu Val Tyr Asp Ala Tyr Leu
195 200 205
Ala Pro Asn Asn Leu Lys Pro Val Val Ala Glu Phe Tyr Gly Ser Lys
210 215 220
Glu Asp Pro Gln Thr Phe Tyr Tyr Ala Val Ala Val Val Lys Lys Asp
225 230 235 240
Ser Gly Phe Gln Met Asn Gln Leu Arg Gly Lys Lys Ser Cys His Thr
245 250 255
Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile Pro Ile Gly Leu Leu Tyr
260 265 270
Cys Asp Leu Pro Glu Pro Arg Lys Pro Leu Glu Lys Ala Val Ala Asn
275 280 285
Phe Phe Ser Gly Ser Cys Ala Pro Cys Ala Asp Gly Thr Asp Phe Pro
290 295 300
Gln Leu Cys Gln Leu Cys Pro Gly Cys Gly Cys Ser Thr Leu Asn Gln
305 310 315 320
Tyr Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu Lys Asp Gly Ala Gly
325 330 335
Asp Val Ala Phe Val Lys His Ser Thr Ile Phe Glu Asn Leu Ala Asn
340 345 350
Lys Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys Leu Asp Asn Thr Arg
355 360 365
Lys Pro Val Asp Glu Tyr Lys Asp Cys His Leu Ala Gln Val Pro Ser
370 375 380
His Thr Val Val Ala Arg Ser Met Gly Gly Lys Glu Asp Leu Ile Trp
385 390 395 400
Glu Leu Leu Asn Gln Ala Gln Glu His Phe Gly Lys Asp Lys Ser Lys
405 410 415
Glu Phe Gln Leu Phe Ser Ser Pro His Gly Lys Asp Leu Leu Phe Lys
420 425 430
Asp Ser Ala His Gly Phe Leu Lys Val Pro Pro Arg Met Asp Ala Lys
435 440 445
Met Tyr Leu Gly Tyr Glu Tyr Val Thr Ala Ile Arg Asn Leu Arg Glu
450 455 460
Gly Thr Cys Pro Glu Ala Pro Thr Asp Glu Cys Lys Pro Val Lys Trp
465 470 475 480
Cys Ala Leu Ser His His Glu Arg Leu Lys Cys Asp Glu Trp Ser Val
485 490 495
Asn Ser Val Gly Lys Ile Glu Cys Val Ser Ala Glu Thr Thr Glu Asp
500 505 510
Cys Ile Ala Lys Ile Met Asn Gly Glu Ala Asp Ala Met Ser Leu Asp
515 520 525
Gly Gly Phe Val Tyr Ile Ala Gly Lys Cys Gly Leu Val Pro Val Leu
530 535 540
Ala Glu Asn Tyr Glu Lys Ser Asp Asn Cys Glu Asp Thr Pro Glu Ala
545 550 555 560
Gly Tyr Phe Ala Val Ala Val Val Lys Lys Ser Ala Ser Asp Leu Thr
565 570 575
Trp Asp Asn Leu Lys Gly Lys Lys Ser Cys His Thr Ala Val Gly Arg
580 585 590
Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Tyr Asn Lys Ile Asn
595 600 605
His Cys Arg Phe Asp Glu Phe Phe Ser Glu Gly Cys Ala Pro Gly Ser
610 615 620
Lys Lys Asp Ser Ser Leu Cys Lys Leu Cys Met Gly Ser Gly Leu Asn
625 630 635 640
Leu Cys Glu Pro Asn Asn Lys Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala
645 650 655
Phe Arg Cys Leu Val Glu Lys Gly Asp Val Ala Phe Val Lys His Gln
660 665 670
Thr Val Pro Gln Asn Thr Gly Gly Lys Asn Pro Asp Pro Trp Ala Lys
675 680 685
Asn Leu Asn Glu Lys Asp Tyr Glu Leu Leu Cys Leu Asp Gly Thr Arg
690 695 700
Lys Pro Val Glu Glu Tyr Ala Asn Cys His Leu Ala Arg Ala Pro Asn
705 710 715 720
His Ala Val Val Thr Arg Lys Asp Lys Glu Ala Cys Val His Lys Ile
725 730 735
Leu Arg Gln Gln Gln His Leu Phe Gly Ser Glu Val Thr Asp Cys Ser
740 745 750
Gly Asn Phe Cys Leu Phe Arg Ser Glu Thr Lys Asp Leu Leu Phe Arg
755 760 765
Asp Asp Thr Val Cys Leu Ala Lys Leu His Asp Arg Asn Thr Tyr Glu
770 775 780
Lys Tyr Leu Gly Glu Glu Tyr Val Lys Ala Val Gly Asn Leu Arg Lys
785 790 795 800
Cys Ser Thr Ser Ser Leu Leu Glu Ala Cys Thr Phe Arg Arg Pro
805 810 815
<210> 2
<211> 2448
<212> DNA
<213>glucagon-like-peptide-1 (the fusion protein F usion protein of of GLP-1 small peptide and transferrins
glucagon-like peptide-1 GLP-1 short peptide and transferrin)
<400> 2
atgcatggag aaggtacttt tacttctgat gtatcttctt atcttgaagg tcaagctgct 60
caagaattca ttgcttggtt ggttgatgga agacatggtg aaggtacttt tacttccgat 120
gtatcttcct atcttgaagg acaagcagca caagaattca ttgcatggtt ggttgatgga 180
cgtcatggcg agggtacttt tacttcagat gtatcttcat atcttgaagg acaagctgca 240
caggaattca ttgcatggtt ggttgatgga agacatgggg aaggtacttt tacttcagac 300
gtatctagtt atcttgaggg acaggcagct caggaattca ttgcatggtt ggtagatgga 360
cgtggaggtg gaggttctgg aggtggaggt tctggaggtg gaggtagtgt acctgataaa 420
actgttagat ggtgtgctgt atctgaacat gaagctacta aatgtcaatc ttttcgtgat 480
catatgaaat ctgttattcc ttctgatgga ccatctgtag cttgtgttaa aaaagcttct 540
tatcttgatt gtattagagc tattgctgct aatgaggctg atgctgttac tttagatgct 600
ggtttagtat atgatgctta tcttgctcct aataacttaa aaccagtagt tgctgaattc 660
tatggatcta aagaagatcc tcaaactttc tattatgctg tagctgtagt taaaaaagat 720
tctggtttcc agatgaatca acttagaggg aaaaaatctt gtcatactgg attaggtcgt 780
tctgctggtt ggaatattcc tattggactt ctttattgtg atcttcctga accaagaaaa 840
ccacttgaaa aagctgttgc taatttcttt tctggttctt gtgctccatg tgctgatgga 900
actgatttcc ctcaactttg tcaactttgt ccaggatgtg gttgttctac tttaaatcaa 960
tatttcggat attctggtgc ttttaaatgt ttaaaagatg gagctggaga tgtagctttc 1020
gttaaacatt ctactatttt cgaaaatctt gctaataagg ctgatagaga tcaatatgaa 1080
cttctttgtc ttgataatac tcgtaaacct gttgatgaat ataaggattg tcatcttgct 1140
caagtaccat ctcatactgt agttgctaga tctatgggag gaaaagaaga tcttatttgg 1200
gaacttctta atcaagctca agaacatttc ggaaaagata aatctaaaga attccaatta 1260
ttttcttctc ctcatggtaa agatcttctt tttaaagatt ctgctcatgg atttttaaaa 1320
gtacctcctc gtatggatgc taaaatgtat cttggttatg aatatgtaac tgctattaga 1380
aatcttcgtg aaggaacttg tcctgaagct ccaactgatg aatgtaaacc agttaaatgg 1440
tgtgctcttt ctcatcatga acgtcttaaa tgtgatgaat ggtctgtaaa ttctgttgga 1500
aaaattgaat gtgtatctgc tgaaactact gaagattgta ttgctaaaat tatgaatggt 1560
gaagctgatg ctatgtctct tgatggaggt ttcgtttata ttgctggaaa atgtggttta 1620
gtacctgttc ttgctgaaaa ttatgaaaaa tctgataatt gtgaagatac tccagaagct 1680
ggatatttcg ctgttgctgt agttaaaaaa tctgcttctg atcttacttg ggataatctt 1740
aaggggaaaa aatcttgcca tactgctgta ggtagaactg ctggatggaa tattcctatg 1800
ggacttcttt ataataagat taatcattgt cgttttgatg aatttttctc tgaaggttgt 1860
gctcctggat ctaaaaaaga ttcttctctt tgtaaattat gtatgggatc tggtcttaat 1920
ctttgtgaac caaataacaa agaaggatat tatggttata ctggagcttt tagatgtctt 1980
gttgaaaaag gagatgttgc attcgttaaa catcaaactg tacctcaaaa tactggagga 2040
aaaaatcctg atccatgggc taaaaatctt aatgagaaag attatgaatt attatgttta 2100
gatggaacta gaaaacctgt agaagaatat gctaattgtc atcttgctag agctccaaat 2160
catgctgtag ttactcgtaa agataaagaa gcttgtgttc ataaaattct tcgtcaacaa 2220
caacatcttt ttggttctga agtaactgat tgttctggaa atttctgttt atttcgttct 2280
gaaactaaag atcttttatt tcgtgatgat actgtttgtt tagctaaact tcatgataga 2340
aatacttatg aaaaatatct tggtgaagaa tatgttaaag ctgttggaaa tcttcgtaaa 2400
tgttctactt cttctcttct tgaagcttgt acttttagac gtccttaa 2448
Claims (10)
1. the fusion protein of glucagon-like-peptide-1 small peptide and transferrins, 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 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 to plant by the codon optimization of the glucagon-like-peptide-1 small peptide and the fusion protein of transferrins
The codon of object preference, nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pGLP-1 is obtained.
6. expression vector or plant as described in claim 3 or 4 is expressing glucagon-like-peptide-1 small peptide and transferrins
Fusion protein or preparation comprising the fusion protein drug in application；The plant is selected from romaine lettuce, spinach, tomato, trailing plants
Fore-telling, 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 the oral preparation of hypoglycemic.
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 the oral preparation of hypoglycemic.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550518.5A CN110218259B (en) | 2019-06-24 | 2019-06-24 | Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules |
| PCT/CN2020/089983 WO2020259111A1 (en) | 2019-06-24 | 2020-05-13 | Application of fabricating oral hypoglycemic capsule from fusion protein of transferrin and plant-produced glucagon-like peptide-1 oligopeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550518.5A CN110218259B (en) | 2019-06-24 | 2019-06-24 | Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110218259A true CN110218259A (en) | 2019-09-10 |
| CN110218259B CN110218259B (en) | 2022-09-16 |
Family
ID=67814502
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910550518.5A Active CN110218259B (en) | 2019-06-24 | 2019-06-24 | Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110218259B (en) |
| WO (1) | WO2020259111A1 (en) |
Cited By (2)
| 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 |
| CN112661862A (en) * | 2020-12-25 | 2021-04-16 | 深圳大学 | Fusion protein and preparation method and application thereof |
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| CN1190438A (en) * | 1995-06-06 | 1998-08-12 | 转移染色体治疗公司 | Chimeric proteins for use in transport of selected substance into cells |
| CN1962695A (en) * | 2005-11-09 | 2007-05-16 | 浙江我武生物科技有限公司 | GLP-1 infusion proteins, their preparation and use |
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| CN102134577A (en) * | 2003-03-28 | 2011-07-27 | 独立行政法人农业生物资源研究所 | Process for producing plant storage organ with high production of recombinant protein and novel recombinant protein |
| AU2005271403A1 (en) * | 2004-08-03 | 2006-02-16 | Biorexis Pharmaceutical Corporation | Combination therapy using transferrin fusion proteins comprising GLP-1 |
| MX348567B (en) * | 2010-06-04 | 2017-06-20 | Tiumbio Co Ltd | Fusion protein having factor vii activity. |
| CN102690352A (en) * | 2011-03-21 | 2012-09-26 | 天津拓飞生物科技有限公司 | Fusion protein containing GLP-1, and pharmaceutical compositions and applications thereof |
| CN110218259B (en) * | 2019-06-24 | 2022-09-16 | 王跃驹 | Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules |
| CN110204620B (en) * | 2019-06-24 | 2022-12-20 | 王跃驹 | Application of plant as host in expression of MGLP fusion protein |
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2019
- 2019-06-24 CN CN201910550518.5A patent/CN110218259B/en active Active
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2020
- 2020-05-13 WO PCT/CN2020/089983 patent/WO2020259111A1/en active Application Filing
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| CN1190438A (en) * | 1995-06-06 | 1998-08-12 | 转移染色体治疗公司 | Chimeric proteins for use in transport of selected substance into cells |
| CN1962695A (en) * | 2005-11-09 | 2007-05-16 | 浙江我武生物科技有限公司 | GLP-1 infusion proteins, their preparation and use |
Non-Patent Citations (3)
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| 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》 * |
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Cited By (2)
| 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 |
| CN112661862A (en) * | 2020-12-25 | 2021-04-16 | 深圳大学 | Fusion protein and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110218259B (en) | 2022-09-16 |
| WO2020259111A1 (en) | 2020-12-30 |
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Effective date of registration: 20230227 Address after: Room 1102, 11/F, Building A7-5, Hanyu Golden Valley, No.7000 Jingshi Road, Jinan Area, China (Shandong) Free Trade Pilot Zone, Jinan, Shandong Province (business premises: Room 607, Block C-1, lattice square, Qilu Innovation Valley) Patentee after: Ruicheng Haihui Biotechnology (Shandong) Co.,Ltd. Address before: Apartment No. 2, 4410 Wetherhill Street, Covallis, Oregon, USA Patentee before: Wang Yueju |