CN110256579A - The application of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule - Google Patents
The application of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule Download PDFInfo
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Abstract
The present invention relates to field of biotechnology, the in particular to application of plant production actrapid monotard and transferrins long-acting oral Jiangtang 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 actrapid monotard and transferrins long-acting oral hypoglycemic
The application of capsule.
Background technique
Diabetes are since insulin absolutely lacks or relative deficiency and target tissue cell (such as fat, liver, skeletal muscle
Deng) comprehensive to the metabolic disorder caused by insulin sensitivity reduction (insulin resistance) characterized by chronic blood glucose level rises height
Disease is closed, can lead to sugar, fat, protein metabolism disorder.Clinically mainly there are insulin-dependent (IDDM, I type) and non-pancreas islet
Plain dependent form (NIDDM, II type) two types.Diabetes have become full generation as a kind of serious non-contagious chronic diseases
One of the great public health problem that various countries, boundary are paid close attention to is the third in global range after cardiovascular and tumor disease
Number killer.With the improvement of living standards, no matter in developed country or in developing country, the disease incidence of diabetes is all year by year
Rise.It is showed in developing country more prominent.Estimate according to WHO, diabetic is more than 1.9 hundred million at present in the whole world, to 2025
Year will be added to 300,000,000.
In the 1980s, people pass through genetic engineering (recombinant DNA) (transgenosis) yeast (brewer's yeast Pichia pastoris
Or Hansenula yeast) or recombination Chinese hamster ovary cell (CHO) give expression to the synthesis actrapid monotard of high-purity, structure and human body
The insulin of itself secretion is the same.Comparing animals insulin, the less generation allergic reaction of actrapid monotard or insulin resistance, institute
Also to be reduced therewith the phenomenon that lipoatrophy；Since actrapid monotard's antibody is few, so injection volume is average than animal insulin
Reduce 30%；The stability of actrapid monotard is higher than animal insulin, and 25 DEG C of room temperature or so room temperature can be reserved for actrapid monotard 4 weeks.?
Onset time, time to peak cannot simulate physiological actrapid monotard on acting duration and secrete mode.It need to be at 30 minutes before the meal
It injects, have higher Nocturnal hypoglycemia risk.
Late 1990s find in the further investigation to actrapid monotard's structure and ingredient, modify peptide chain:
Using technique for gene engineering, change the combination of amino acids at certain positions on insulin peptide chain；Change isoelectric point；It is strong to increase by six aggressiveness
Degree；Zinc ion is substituted with cobalt ions；Increase fatty acid chain in the molecule, the combination of increasing and albumin is possible to change it
Physical and chemical and biological property, so as to develop the insulin analog for being more suitable for Human Physiology needs
(insulinsimilitude).Insulin analog can be close to meal and use, insulin or Semilente Insulin when also referred to as eating.
Although insulin has many advantages, such as in treatment diabetes, its property and people due to polypeptide drug itself
The various barriers that body generates it, conventional administration by way of always with injection based on.The present invention is by actrapid monotard and transferrins
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 provides answering for a kind of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule
With.The present invention carries out structure of modification and modification to the active peptides with blood sugar reducing function, inhale its acquisition can by enteron aisle
The characteristic of effective treatment concentration is received and reached in vivo, and produces the active material by plant.The present invention is outstanding using plant
It is the efficient platform technology that romaine lettuce is produced as recombinant protein, expresses the fusion protein sequence of actrapid monotard and transferrins
Column.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 actrapid monotard 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 also provides the nucleotide for encoding fusion protein as described in claim 1, 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 the basis of the studies above, the present invention also provides expression vectors, including the nucleotide and load to be transformed
Body.
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 favorite plant by the codon optimization of the actrapid monotard and the fusion protein of transferrins
Codon, nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pInTf is obtained.
The present invention also provides the expression vectors or plant in the fusion protein for expressing actrapid monotard and transferrins
Or preparation includes the application in the drug of the fusion protein；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 drug is the oral preparation of hypoglycemic.
On the basis of the studies above, the present invention also provides hosts, convert the plant for having the expression vector or micro- life
Object；The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The organ of the plant selects
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.
A kind of method the present invention also provides plant as host expresses actrapid monotard and the fusion protein of transferrins,
By expression vector biolistic bombardment blade, regeneration plant is obtained after expressing in plant chloroplast, by plant leaf blade
Freeze-drying is crushed, is extracted, and obtains the fusion protein of actrapid monotard and transferrins.
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 actrapid monotard 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.
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
It is bitter.Romaine lettuce does not contain plant noxious material, and this product is not required to protein purification process, can greatly shorten production cycle and life
Produce 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 the fusion protein sequence of romaine lettuce system large-scale production actrapid monotard and transferrins.
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 pInTf schematic diagram；
Fig. 2 shows western-blot result.
Specific embodiment
The invention discloses the application of a kind of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule, abilities
Field technique personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replaces
Change and change apparent to those skilled in the art, they are considered as being included in the present invention.Side of the invention
Method and application be described by preferred embodiment, related personnel obviously can not depart from the content of present invention, spirit and
To method described herein and application is modified or appropriate changes and combinations in range, 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, expresses the fusion protein sequence of actrapid monotard and transferrins.And it is made oral
Jiangtang capsule.
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 expression actrapid monotard and transferrins fusion protein sequence application.
Preferably, the plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The device of the plant
Official is selected from seed, leaf, rhizome or whole plant.The present invention also provides a kind of expression vector, including actrapid monotard and turn iron egg
White fusion protein sequence nucleotide sequence and carrier.
In some specific embodiments of the invention, the fusion protein stream cipher of the actrapid monotard and transferrins
Son is optimized for the codon of favorite plant.
In some specific embodiments of the invention, the actrapid monotard of the optimization and the fusion protein sequence of transferrins
Column are as shown in SEQ ID No.1；The nucleotide sequence of the fusion protein sequence of the actrapid monotard and transferrins of the optimization is such 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: being the password of favorite plant by the codon optimization of actrapid monotard and the fusion protein sequence of transferrins
Son；
Step 2: gene chemical synthesis being carried out by Jin Sirui and is cloned into pUC57 carrier, pInTf cloning vector is obtained
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by actrapid monotard and transferrins
Fusion protein sequence amino acid sequence utilize anti-translation software (https: //www.ebi.ac.uk/Tools/st/emboss_
Backtranseq/ nucleotide sequence) is obtained, and is the codon of favorite plant by its codon optimization, by Jin Sirui company
(Nanjing, China) synthesis.And it is grand into pUC57 carrier by golden Stryker, it obtains pInTf carrier (Fig. 1).
The present invention also provides the expression vectors in the fusion protein sequence of expression actrapid monotard and transferrins
Application.
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.
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
It is bitter.Romaine lettuce does not contain plant noxious material, and this product is not required to protein purification process, can greatly shorten production cycle and life
Produce 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 the fusion protein sequence of romaine lettuce system large-scale production actrapid monotard and transferrins.
Original used in the application of plant production actrapid monotard provided by the invention and transferrins long-acting oral Jiangtang capsule
Material and reagent are available on the market.
Below with reference to embodiment, the present invention is further explained:
The building of 1 chloroplast expression vector of embodiment
For the high efficient expression by foreign protein in plant, by the fusion protein sequence ammonia of actrapid monotard and transferrins
Base acid sequence utilizes anti-translation software
(https: //www.ebi.ac.uk/Tools/st/emboss_backtranseq/) obtains nucleotide sequence, and
It 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).
6 Western blot testing goal protein expression situation of embodiment
Vegetable protein is extracted using liquid nitrogen grinding, denatured lysis, cracking 5 × sample-loading buffer of supernatant (is added using preceding
Enter beta -mercaptoethanol to it is final concentration of 5%) in 4:1 ratio mixing (such as 200 μ l protein cleavage supernatants and 50 μ 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, the results showed that target protein normal expression in romaine lettuce is shown in figure
2。
The detection of the fusion protein sequence active of 7 actrapid monotard of embodiment 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 actrapid monotard made from embodiment 5 and transferrins) and without in two kinds of experiment capsules of blood sugar reducing proteins
One kind is done and is repeated for the first time.Dog is grouped at random again, receives another different experimental diet, does second of repetition.It repeats
I and II are at least for 2 weeks, detect 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) (fusion protein of actrapid monotard and transferrins that the present invention obtains), and be free of hypoglycemic
One of two kinds of experiment capsules of albumen, receive identical experimental diet.Continuous feeding 10 days is seen in fact after each feeding
It examines, needs to be observed continuously daily 6 hours or more, do not see that mouse is in excitatory state or holddown, it is not existing out
Phenomena such as slow is moved, situations such as diarrhea does not also occur.Prove the fusion protein oral administration safety of actrapid monotard and transferrins
It is high.
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 plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule
<130> MP1907675
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 804
<212> PRT
<213>fusion protein (the Fusion protein of human insulin and of actrapid monotard and transferrins
transferrin)
<400> 1
Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu
1 5 10 15
Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly
20 25 30
Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe
35 40 45
Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val Gly
50 55 60
Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gln Pro Leu
65 70 75 80
Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile Val Glu Gln Cys Cys
85 90 95
Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Pro Asp
115 120 125
Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu Ala Thr Lys Cys
130 135 140
Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro Ser Asp Gly Pro
145 150 155 160
Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp Cys Ile Arg Ala
165 170 175
Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp Ala Gly Leu Val
180 185 190
Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro Val Val Ala Glu
195 200 205
Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr Tyr Ala Val Ala
210 215 220
Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln Leu Arg Gly Lys
225 230 235 240
Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly Trp Asn Ile Pro
245 250 255
Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg Lys Pro Leu Glu
260 265 270
Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala Pro Cys Ala Asp
275 280 285
Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro Gly Cys Gly Cys
290 295 300
Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala Phe Lys Cys Leu
305 310 315 320
Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His Ser Thr Ile Phe
325 330 335
Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr Glu Leu Leu Cys
340 345 350
Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys Asp Cys His Leu
355 360 365
Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser Met Gly Gly Lys
370 375 380
Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln Glu His Phe Gly
385 390 395 400
Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser Pro His Gly Lys
405 410 415
Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu Lys Val Pro Pro
420 425 430
Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr Val Thr Ala Ile
435 440 445
Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro Thr Asp Glu Cys
450 455 460
Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu Arg Leu Lys Cys
465 470 475 480
Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu Cys Val Ser Ala
485 490 495
Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn Gly Glu Ala Asp
500 505 510
Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala Gly Lys Cys Gly
515 520 525
Leu Val Pro Val Leu Ala Glu Asn Tyr Glu Lys Ser Asp Asn Cys Glu
530 535 540
Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val Val Lys Lys Ser
545 550 555 560
Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys Lys Ser Cys His
565 570 575
Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu
580 585 590
Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe Phe Ser Glu Gly
595 600 605
Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys Lys Leu Cys Met
610 615 620
Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys Glu Gly Tyr Tyr
625 630 635 640
Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys Gly Asp Val Ala
645 650 655
Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly Gly Lys Asn Pro
660 665 670
Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr Glu Leu Leu Cys
675 680 685
Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala Asn Cys His Leu
690 695 700
Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys Asp Lys Glu Ala
705 710 715 720
Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu Phe Gly Ser Glu
725 730 735
Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg Ser Glu Thr Lys
740 745 750
Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala Lys Leu His Asp
755 760 765
Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr Val Lys Ala Val
770 775 780
Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu Glu Ala Cys Thr
785 790 795 800
Phe Arg Arg Pro
<210> 2
<211> 2412
<212> DNA
<213>fusion protein (the Fusion protein of human insulin and of actrapid monotard and transferrins
transferrin)
<400> 2
atggctttat ggatgagact tcttcctctt cttgctcttc ttgctttatg gggacctgat 60
cctgctgctg cttttgtaaa tcaacatctt tgtggttctc atcttgttga agctttatat 120
cttgtatgtg gtgaacgagg atttttctat actcctaaaa caagacgaga agctgaagat 180
cttcaagtag gacaagttga attaggtgga ggacctggtg ctggatcttt acaacctctt 240
gctcttgaag gatctcttca aaaaagaggt attgtagaac aatgttgtac ttctatttgt 300
tctctttatc aacttgaaaa ttattgtaat ggtggaggtg gatctggtgg aggtggatct 360
ggtggaggtg gaagtgtacc tgataaaact gttagatggt gtgctgtatc tgaacatgaa 420
gctacaaaat gtcaatcttt tcgagatcat atgaaatctg ttattccttc tgatggacct 480
tctgtagctt gtgttaaaaa agcttcttat cttgattgta ttcgagctat tgctgctaat 540
gaggctgatg ctgttacatt agatgctgga ttagtatatg atgcttatct tgctcctaat 600
aacttaaaac ctgtagttgc tgaattctat ggttctaaag aagatcctca aactttctat 660
tatgctgtag ctgtagttaa aaaagattct ggattccaga tgaatcaact tagagggaaa 720
aaatcttgtc atacaggttt aggacgatct gctggatgga atattcctat tggtcttctt 780
tattgtgatc ttcctgaacc tagaaaacct cttgaaaaag ctgttgctaa tttcttttct 840
ggatcttgtg ctccttgtgc tgatggtact gatttccctc aactttgtca actttgtcct 900
ggttgtggat gttctacatt aaatcaatat ttcggttatt ctggagcttt taaatgttta 960
aaagatggtg ctggagatgt agctttcgtt aaacattcta ctattttcga aaatcttgct 1020
aataaggctg atagagatca atatgaactt ctttgtcttg ataatactcg aaaacctgtt 1080
gatgaatata aggattgtca tcttgctcaa gtaccttctc atacagtagt tgctagatct 1140
atgggaggaa aagaagatct tatttgggaa cttcttaatc aagctcaaga acatttcgga 1200
aaagataaat ctaaagaatt ccaattattt tcttctcctc atggaaaaga tcttcttttt 1260
aaagattctg ctcatggatt tttaaaagta cctcctcgta tggatgctaa aatgtatctt 1320
ggatatgaat atgtaactgc tattagaaat cttcgagaag gtacttgtcc tgaagctcct 1380
acagatgaat gtaaacctgt taaatggtgt gctctttctc atcatgaacg acttaaatgt 1440
gatgaatggt ctgtaaattc tgttggaaaa attgaatgtg tatctgctga aactacagaa 1500
gattgtattg ctaaaattat gaatggtgaa gctgatgcta tgtctcttga tggtggattc 1560
gtttatattg ctggtaaatg tggattagta cctgttcttg ctgaaaatta tgaaaaatct 1620
gataattgtg aagatactcc tgaagctgga tatttcgctg ttgctgtagt taaaaaatct 1680
gcttctgatc ttacatggga taatcttaag gggaaaaaat cttgccatac tgctgtagga 1740
agaacagctg gttggaatat tcctatgggt cttctttata ataagattaa tcattgtcga 1800
tttgatgaat ttttctctga aggatgtgct cctggatcta aaaaagattc ttctctttgt 1860
aaattatgta tgggttctgg acttaatctt tgtgaaccta ataacaaaga aggatattat 1920
ggatatactg gtgcttttag atgtcttgtt gaaaaaggag atgttgcatt cgttaaacat 1980
caaactgtac ctcaaaatac aggtggaaaa aatcctgatc cttgggctaa aaatcttaat 2040
gagaaagatt atgaattatt atgtttagat ggtactagaa aacctgtaga agaatatgct 2100
aattgtcatc ttgctagagc tcctaatcat gctgtagtta cacgaaaaga taaagaagct 2160
tgtgttcata aaattcttcg acaacaacaa catctttttg gatctgaagt aactgattgt 2220
tctggtaatt tctgtttatt tcgatctgaa actaaagatc ttttatttcg agatgataca 2280
gtttgtttag ctaaacttca tgatagaaat acatatgaaa aatatcttgg agaagaatat 2340
gttaaagctg ttggtaatct tcgaaaatgt tctacttctt ctcttcttga agcttgtact 2400
tttagaagac ct 2412
Claims (10)
1. the fusion protein of actrapid monotard 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 ID2；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 password of favorite plant by the codon optimization of the actrapid monotard and the fusion protein of transferrins
Son, nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pInTf is obtained.
6. expression vector as described in claim 3 or 4 or plant expression actrapid monotard and transferrins fusion protein or
Preparation includes the application in the drug of the fusion protein；The plant be selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn,
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 (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550526.XA CN110256579A (en) | 2019-06-24 | 2019-06-24 | The application of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550526.XA CN110256579A (en) | 2019-06-24 | 2019-06-24 | The application of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110256579A true CN110256579A (en) | 2019-09-20 |
Family
ID=67920984
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910550526.XA Pending CN110256579A (en) | 2019-06-24 | 2019-06-24 | The application of plant production actrapid monotard and transferrins long-acting oral Jiangtang capsule |
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| CN (1) | CN110256579A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591417A (en) * | 2022-04-22 | 2022-06-07 | 四川大学 | Human single-chain insulin analogue and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2019
- 2019-06-24 CN CN201910550526.XA patent/CN110256579A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| TRACEY A. RUHLMAN: "Plastid Transformation in Lettuce ( Lactuca sativa L.) by Biolistic DNA Delivery", 《METHODS IN MOLECULAR BIOLOGY》 * |
| YU-SHENG CHEN等: "Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec", 《INT. J. MOL. SCI.》 * |
| 无: "NCBI Reference Sequence: NP_000198.1", 《GENEBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114591417A (en) * | 2022-04-22 | 2022-06-07 | 四川大学 | Human single-chain insulin analogue and application thereof |
| CN114591417B (en) * | 2022-04-22 | 2023-04-25 | 四川大学 | Human single chain insulin analogues and uses thereof |
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Application publication date: 20190920 |