CN110256581A - Plant production takes orally the application of the fusion protein Jiangtang capsule of Suo Malutai (Semaglutide) and Lumbrokinase - Google Patents
Plant production takes orally the application of the fusion protein Jiangtang capsule of Suo Malutai (Semaglutide) and Lumbrokinase Download PDFInfo
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Abstract
The present invention relates to biomedicine technical fields, the in particular to application of plant production oral Suo Malutai (Semaglutide) and Lumbrokinase 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 biomedicine technical field, in particular to plant production takes orally Suo Malutai (Semaglutide)
With the application of the fusion protein Jiangtang capsule of Lumbrokinase.
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.
Summary of the invention
In view of this, the present invention provides the applications that plant production takes orally Du Lalu peptide Yu Nattokinase Jiangtang capsule.This
The efficient platform technology produced using plant especially romaine lettuce as recombinant protein is invented, Suo Malutai is expressed
The fusion protein of Semaglutide and Lumbrokinase.And orally-taken blood sugar reducing capsule is made.Therefore it provides a kind of diabetes for the treatment of
Oral preparation has important practical significance.
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 Suo Malutai (Semaglutide) and Lumbrokinase, 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.
In some specific embodiments of the invention, it is the multiple be 2,3,4,5,6,7,8,9
A, 10,11,12,13,14,15 or 16,17,18,19,20,21,22,23,24
A, 25,26,27,28,29,30,31 or 32.
The present invention also 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.
In some specific embodiments of the invention, it is the multiple be 2,3,4,5,6,7,8,9
A, 10,11,12,13,14,15 or 16,17,18,19,20,21,22,23,24
A, 25,26,27,28,29,30,31 or 32.
The present invention also provides expression vectors, including the nucleotide and carrier to be transformed.
In some specific embodiments of the invention, the carrier to be transformed is chloroplast expression vector.
On the basis of the studies above, the present invention also provides the construction methods of the expression vector, including walk as follows
It is rapid:
Step 1: being by the Suo Malutai (Semaglutide) and the codon optimization of the fusion protein of Lumbrokinase respectively
The codon of favorite plant, nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pUC57-sem (Suo Malu peptide and earthworm are obtained
The fusion protein of kinases).
The present invention also provides the expression vectors or plant in expression Suo Malutai (Semaglutide) and Lumbrokinase
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.
In some specific embodiments of the invention, the drug is oral hypoglycemic agents.
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 oral hypoglycemic agents.
In addition, the present invention also provides a kind of plants as host expresses Suo Malutai (Semaglutide) and Lumbrokinase
The method of fusion protein expression vector biolistic bombardment blade is obtained again after expressing in plant chloroplast
Plant leaf blade is lyophilized crushed filling capsule, Jiangtang capsule is made by raw plant.
In some specific embodiments of the invention, the biolistic bombardment includes the following steps:
Step 1: preparing conversion carrier；
Step 2: preparing particle bullet；
Step 3: biolistic bombardment；
Step 4: being cultivated after conversion, be regenerated as plant.
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.
Suo Malutai (Semaglutide) and Lumbrokinase amalgamation and expression, same realize are being administered orally, are being mitigated by the present invention
Sufferer long term frequent injects bring pain.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows carrier pUC57-sem schematic diagram；
Fig. 2 shows western-blot result；Wherein, swimming lane 1 shows non-express plant；Swimming lane 2 shows that romaine lettuce produces Suo Malutai
(Semaglutide) with Lumbrokinase fusion protein.
Specific embodiment
The invention discloses the application that a kind of plant production takes orally Du Lalu peptide and Nattokinase Jiangtang capsule, this field skills
Art personnel can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar replacements and
Change apparent to those skilled in the art, they are considered as being included in the present invention.Method of the invention and
Using being described by preferred embodiment, related personnel can obviously not depart from the content of present invention, spirit and scope
It is interior that method described herein and application are modified or appropriate changes and combinations, carry out implementation and application the technology of the present invention.
The present invention provides plant as host in the fusion protein of expression Suo Malutai Semaglutide and Lumbrokinase
Using.Preferably, the plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn and soybean, wheat or tobacco；The plant
The organ of object is selected from seed, leaf, rhizome or whole plant.The present invention also provides a kind of expression vectors, including Suo Malutai
The fusion protein sequence and carrier of Semaglutide and Lumbrokinase.
In some specific embodiments of the invention, the Suo Malutai Semaglutide merges egg with Lumbrokinase
White codon optimization is the codon of favorite plant.
In some specific embodiments of the invention, the Suo Malutai Semaglutide of the optimization and Lumbrokinase
Fusion protein sequence is as shown in SEQ ID No.1；The Suo Malutai Semaglutide of the optimization and the fusion protein of Lumbrokinase
Nucleotide sequence 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 favorite plant by the codon optimization of Suo Malutai Semaglutide and the fusion protein of Lumbrokinase
Codon；
Step 2: gene chemical synthesis being carried out by Jin Sirui and is cloned into pUC57 carrier, pUC57-sem cloning vector is obtained；
Specifically, in order to provide high efficient expression of the foreign protein in plant, the present invention is by Suo Malutai Semaglutide
Anti- translation software (https: //www.ebi.ac.uk/Tools/st/ is utilized with the fusion protein amino acid sequence of Lumbrokinase
Emboss_backtranseq/ nucleotide sequence) is obtained, and is the codon of favorite plant, You Jinsi by its codon optimization
Auspicious company (Nanjing, China) synthesis.And it is grand into pUC57 carrier by golden Stryker, it obtains pUC57-sem carrier (Fig. 1).
The present invention also provides the expression vectors to merge egg in expression Suo Malutai Semaglutide and Lumbrokinase
Application in white.
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 carries out structure of modification and modification to the active peptides with blood sugar reducing function, make its acquisition can by enteron aisle into
Row absorbs and reaches the characteristic of effective treatment concentration in vivo, and produces the active material present invention by plant and pass through experiment
It was found that botanical system especially romaine lettuce system be it is more economical, efficiently express platform, chloroplaset can efficient expression activity
Albumen.Due to romaine lettuce be easy to grow and can commercial mass production, than other plants, such as tobacco is easier to obtain simultaneously
And it is cheaper, and due to not needing complicated special producing equipment, cost can significantly reduce.In conclusion the present invention can be with
Utilize the fusion protein of romaine lettuce system large-scale production Suo Malutai Semaglutide and Lumbrokinase.
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.
Plant provided by the invention is as host in the fusion protein of expression Suo Malutai Semaglutide and Lumbrokinase
Application in raw materials used and reagent be 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 amino of Semaglutide and Lumbrokinase
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.
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 5 × loadings of l delay
Fliud flushing mixing), it mixes, 95 DEG C of heating 6min, while handling negative control and positive control；Electrophoretic voltage spacer gel 80V, separation
Glue 120V is run after destination protein to separation gel middle position, stops electrophoresis, is recycled lower slot electrophoresis liquid, is dismantled electrophoretic apparatus, press
According to cathode (black), sponge, filter paper, gel, pvdf membrane (1 × transfer is soaked in after being washed in advance with methanol activation 15s, ddH2O
In buffer) or NC film (being not required to activate), filter paper, sponge, anode (transparent) sequence place, exhaust bubble after assemble, be put into electricity
Swimming slot (note black corresponds to electrophoresis tank black and is put on one side), fills it up with transfering buffering liquid, entire electrophoresis tank is put into ice water mixed liquor
In, 90V electrophoresis 1.0h；5% skimmed milk power (confining liquid) is prepared at the end of electrophoresis is fast, and the film after transfer is put into room in confining liquid
Temperature closing at least 1h, 4 DEG C of incubation primary antibodies are overnight (primary antibody is diluted in 5% skimmed milk power, thinner ratio reference book)；It uses
PBST or TBST washs 15min × 3 time, and incubation at room temperature 1~2h of secondary antibody, PBST or TBST are washed 15min × 3 time, tried using DAB
Agent box develops the color, and takes pictures, and gel shows that albumen size is 32kDa, with expected Suo Malutai Semaglutide and Lumbrokinase
Fusion protein molecule amount it is in the same size, it was demonstrated that we successfully express and are purified purpose fusion protein.
The fusion protein Activity determination of 7 Semaglutide of embodiment and Lumbrokinase
After continuing seven weeks stationary phases, dog is randomly divided into three treatment groups, every group 10, receives contain hypoglycemic respectively
Albumen (fusion protein of Suo Malutai Semaglutide and Lumbrokinase made from the embodiment of the present invention 5) and be free of blood sugar reducing proteins
One of two kinds of experiment capsules commercially use Suo Malutai Semaglutide (according to weight feeding 500ng/g) right as the positive
According to dog is grouped at random, receives identical experimental diet and 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.Repeat 3
Secondary, the experimental data of collection is average value ± SD, and positive controls are business Semaglutide.
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)；
Experimental result shows, compared with business Suo Malutai Semaglutide, containing Suo Malutai Semaglutide and
The fusion protein capsule of Lumbrokinase significantly reduces dog blood-sugar content (P < 0.001).After 2 hours, 3 fusion protein glue are taken orally
Capsule dog blood sugar concentration is detected as 3.4 ± 0.018mmol/L, and takes orally the dog blood glucose of 3 business Suo Malutai Semaglutide
Concentration is 4.1 ± 0.011mmol/L.Illustrate that fusion protein uses Suo Malutai Semaglutide more resistant to gastric acid compared with business
It decomposes, explains blood glucose to be easily accessible in blood.
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 Suo Malutai Semaglutide and Lumbrokinase that the present invention obtains), quotient
Industry Suo Malutai Semaglutid (according to weight feeding 500ng/g) is as positive control, and is free of two kinds of realities of blood sugar reducing proteins
One of capsule is tested, identical experimental diet is received.Continuous feeding 10 days is observed after each feeding in fact, is needed daily
It is observed continuously 6 hours or more, does not see that mouse is in excitatory state or holddown, do not occur being slow in action etc. existing
As also there is not situations such as diarrhea.Prove the fusion protein capsule oral safety of Suo Malutai Semaglutide and Lumbrokinase
Property 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>plant production takes orally the application of the fusion protein Jiangtang capsule of Suo Malutai (Semaglutide) and Lumbrokinase
<130> MP1902781
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 301
<212> PRT
<213>Suo Malutai (the fusion protein F usion protein of Semaglutide of Semaglutide and Lumbrokinase
and lumbrokinase)
<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 Lys Glu Phe Ile Ala Trp Leu Val Arg 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 Gly Ile Val
50 55 60
Gly Gly Ile Glu Ala Arg Pro Tyr Glu Phe Pro Trp Gln Val Ser Val
65 70 75 80
Arg Arg Lys Ser Ser Asp Ser His Phe Cys Gly Gly Ser Ile Ile Asn
85 90 95
Asp Arg Trp Val Val Cys Ala Ala His Cys Met Gln Gly Glu Ser Pro
100 105 110
Ala Leu Val Ser Leu Val Val Gly Glu His Asp Ser Ser Ala Ala Ser
115 120 125
Thr Val Arg Gln Thr His Asp Val Asp Ser Ile Phe Val His Glu Asp
130 135 140
Tyr Asn Gly Asn Thr Phe Glu Asn Asp Val Ser Val Ile Lys Thr Val
145 150 155 160
Asn Ala Ile Ala Ile Asp Ile Asn Val Gly Pro Ile Cys Ala Pro Asp
165 170 175
Pro Ala Asn Asp Tyr Val Tyr Arg Lys Ser Gln Cys Ser Gly Trp Gly
180 185 190
Thr Ile Asn Ser Gly Gly Val Cys Cys Pro Asn Val Leu Arg Tyr Val
195 200 205
Thr Leu Asn Val Thr Thr Asn Ala Phe Cys Asp Asp Ile Tyr Ser Pro
210 215 220
Leu Tyr Thr Ile Thr Ser Asp Met Ile Cys Ala Thr Asp Asn Thr Gly
225 230 235 240
Gln Asn Glu Arg Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser
245 250 255
Val Lys Asp Gly Ser Gly Ile Phe Ser Leu Ile Gly Ile Val Ser Trp
260 265 270
Gly Ile Gly Cys Ala Ser Gly Tyr Pro Gly Val Tyr Ala Arg Val Gly
275 280 285
Ser Gln Thr Gly Trp Ile Thr Asp Ile Ile Thr Asn Asn
290 295 300
<210> 2
<211> 906
<212> DNA
<213>Suo Malutai (the fusion protein F usion protein of Semaglutide of Semaglutide and Lumbrokinase
and lumbrokinase)
<400> 2
atgcacggag aaggtacttt tacttctgat gtttcttctt atcttgaagg acaagctgct 60
aaagaattca ttgcttggtt agttagaggt cgtcatggtg aaggtacttt tacttccgat 120
gtatcttcct atcttgaagg tcaagctgct caagaattca ttgcatggtt ggttgatgga 180
agaggtattg ttggaggaat tgaagctcgt ccttatgaat ttccatggca agtatctgtt 240
agacgtaaat cttctgattc tcatttctgt ggaggttcta ttattaatga tagatgggta 300
gtttgtgctg ctcattgtat gcaaggagaa tctcctgctt tagtttcttt agtagttggt 360
gaacatgatt cttctgctgc ttctactgta agacaaactc atgatgttga ttctattttc 420
gtacatgaag attataatgg taatactttt gaaaatgatg tatctgttat taaaactgtt 480
aatgctattg ctattgatat taatgtaggt ccaatttgtg ctcctgatcc agctaatgat 540
tatgtttata gaaaatctca atgttctgga tggggtacta ttaattctgg aggtgtatgt 600
tgtcctaatg tacttcgtta tgttactctt aatgtaacta ctaatgcttt ctgtgatgat 660
atctattctc cattatatac tattacttct gatatgattt gtgctactga taatactgga 720
caaaatgaga gagattcttg tcaaggagat tctggaggtc ctctttctgt taaagatgga 780
tctggtattt ttagtcttat tggaattgta tcttggggaa ttggttgtgc ttctggatat 840
ccaggtgttt atgctcgtgt aggatctcaa actggttgga ttactgatat tattactaat 900
aactaa 906
Claims (10)
1. the fusion protein of Suo Malutai and Lumbrokinase, 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: it is respectively the codon of favorite plant by the codon optimization of the Suo Malutai and the fusion protein of Lumbrokinase,
Its nucleotide sequence is as shown in SEQ ID No.2；
Step 2: the nucleotide sequence being cloned into pUC57 carrier, pUC57-sem is obtained.
6. expression vector as described in claim 3 or 4 or plant are in the fusion protein or system of expression Suo Malutai and Lumbrokinase
Application in the standby drug comprising the fusion protein；The plant is selected from romaine lettuce, spinach, tomato, radish, Chinese cabbage, corn, big
Beans, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550611.6A CN110256581B (en) | 2019-06-24 | 2019-06-24 | Application of plant in producing oral somaglutide and lumbrokinase fusion protein hypoglycemic capsule |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910550611.6A CN110256581B (en) | 2019-06-24 | 2019-06-24 | Application of plant in producing oral somaglutide and lumbrokinase fusion protein hypoglycemic capsule |
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| CN110256581B CN110256581B (en) | 2022-08-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107083398A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in the expression antibody of PD 1 and/or PD L1 antibody |
| CN108192910A (en) * | 2017-09-19 | 2018-06-22 | 陈玉皎 | Cultivate hypoglycemic and drop thrombus genetically modified plants |
-
2019
- 2019-06-24 CN CN201910550611.6A patent/CN110256581B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN107083398A (en) * | 2017-06-16 | 2017-08-22 | 深圳惠升生物科技有限公司 | Application of the plant as host in the expression antibody of PD 1 and/or PD L1 antibody |
| CN108192910A (en) * | 2017-09-19 | 2018-06-22 | 陈玉皎 | Cultivate hypoglycemic and drop thrombus genetically modified plants |
Non-Patent Citations (3)
| Title |
|---|
| FANGFANG XU等: "Bioactivity of a modified human Glucagon-like peptide-1", 《PLOS ONE》 * |
| JINPEI ZHOU等: "Synthesis and Bioactivity Evaluation of Dipeptidyl Peptidase IV Resistant Glucagon-like Peptide-1 Analogues", 《PROTEIN & PEPTIDE LETTERS》 * |
| TRACEY A. RUHLMAN: "Plastid Transformation in Lettuce ( Lactuca sativa L.) by Biolistic DNA Delivery", 《METHODS IN MOLECULAR BIOLOGY》 * |
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|---|---|
| CN110256581B (en) | 2022-08-05 |
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Effective date of registration: 20230222 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 |