CN110256581B

CN110256581B - Application of plant in producing oral somaglutide and lumbrokinase fusion protein hypoglycemic capsule

Info

Publication number: CN110256581B
Application number: CN201910550611.6A
Authority: CN
Inventors: 王跃驹
Original assignee: Individual
Current assignee: Ruicheng Haihui Biotechnology (Shandong) Co.,Ltd.
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2022-08-05
Anticipated expiration: 2039-06-24
Also published as: CN110256581A

Abstract

The invention relates to the technical field of biological medicine, in particular to application of a plant to the production of oral somaglutide and lumbrokinase hypoglycemic capsules. The present invention utilizes plants such as lettuce as an efficient expression platform for recombinant protein production, and utilizes a simple and efficient expression system to produce bioactive substances. The leaves from which the active substance is produced are subsequently freeze-dried to form capsules. The capsule can be preserved at room temperature while maintaining biological activity. And determining the successful expression of the hypoglycemic active protein by using a Western Blot protein hybridization method. The biological activity test result shows that the blood sugar reducing capsule produced by the platform technology obviously reduces the blood sugar concentration of the dog blood.

Description

Application of plant in producing oral somaglutide and lumbrokinase fusion protein hypoglycemic capsule

Technical Field

The invention relates to the technical field of biological medicine, in particular to application of a plant to the production of an oral somaglutide and lumbrokinase fusion protein hypoglycemic capsule.

Background

Diabetes is a common disease or frequently encountered disease characterized by chronic hyperglycemia, and is a disorder of metabolism of sugar, fat and protein caused by defective secretion or action of insulin in vivo or by the presence of both of them. Clinically, there are two major types, insulin-dependent (IDDM, type I) and non-insulin-dependent (NIDDM, type II). With the increase in living standard, the incidence of diabetes has increased year by year both in developed and developing countries. Diabetes, a serious non-infectious chronic disease, has now become one of the major public health problems of great concern in countries around the world, and is number three killer following cardiovascular and neoplastic diseases worldwide. From the data published by the world health organization, there are only about 3000 million diabetics worldwide in 1995, and have increased to 1.35 billion in 1997, and 3 billion type II diabetics in 2030, with asia and africa being the areas where patients are most rapidly amplified. The traditional treatment modalities for type II diabetics generally follow a stepwise treatment with dietary controls, oral antidiabetic drugs and exogenous insulin. However, there are still many important problems to be solved in the field of diabetes treatment, and there are some side effects and limitations.

Glucagon-like peptide-1 (GLP-1) is an incretin secreted by endocrine cells in the intestinal tract, is a post-translational product of the Glucagon protogene, and exists in various forms in the body. It has the following physiological effects: acting on islet beta cells in a glucose-dependent manner, promoting the transcription of insulin genes, and increasing the biosynthesis and secretion of insulin; stimulating the proliferation and differentiation of beta cells, inhibiting beta cell apoptosis, increasing the number of pancreatic beta cells, inhibiting glucagon secretion, suppressing appetite and ingestion, delaying gastric content emptying, etc. These functions are all beneficial in lowering postprandial blood glucose and maintaining blood glucose at a constant level.

Although natural GLP-1 has a plurality of advantages in treating diabetes, the half-life period in vivo is only about 2 minutes, which limits the direct application of the natural GLP-1 in clinic. After some amino acids in the natural GLP-1 are mutated, the half-life period of the natural GLP-1 can be prolonged under the condition of ensuring the activity of the natural GLP-1, and the normal blood sugar level can be kept by once-weekly administration. The related products on the market at present include Liraglutide, Dulaglutide, Semaglutide and the like. Due to the nature of polypeptide drugs and the various barriers that the human body creates, the conventional administration route has been mainly injection.

Disclosure of Invention

In view of the above, the invention provides an application of plants in producing oral dolabrin and nattokinase hypoglycemic capsules. The invention uses plants, especially lettuce, as an efficient platform technology for recombinant protein production to express the fusion protein of the thaumatin Semaglutide and lumbrokinase. And making into oral blood sugar lowering capsule. Therefore, the oral preparation for treating diabetes has important practical significance.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a fusion protein of thaumautta (Semaglutide) and lumbrokinase, which comprises the following components:

(I) an amino acid sequence shown as SEQ ID No. 1; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 80% homology with the sequence of (I) or (II).

In some embodiments of the invention, the plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32.

The invention also provides the nucleotide encoding the fusion protein, having

(I) a nucleotide sequence shown as SEQ ID No. 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID 2; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or

(V) a nucleotide sequence having at least 80% homology with the nucleotide sequence of (I), (II), (III) or (IV).

The invention also provides an expression vector, which comprises the nucleotide and a vector to be transformed.

In some embodiments of the invention, the vector to be transformed is a chloroplast expression vector.

On the basis of the research, the invention also provides a construction method of the expression vector, which comprises the following steps:

step 1: respectively optimizing the codons of the fusion protein of the somaltulide (Semaglutide) and the lumbrokinase into codons preferred by plants, wherein the nucleotide sequence of the fusion protein is shown as SEQ ID No. 2;

step 2: the nucleotide sequence was cloned into a pUC57 vector to obtain pUC57-sem (a fusion protein of somatid and lumbrokinase).

The invention also provides application of the expression vector or the plant in expressing the fusion protein of the Somatoxylum pratense (Semaglutide) and the lumbrokinase or preparing a medicament containing the fusion protein; the plant is selected from lettuce, spinach, tomato, radish, Chinese cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

In some embodiments of the invention, the medicament is an oral hypoglycemic formulation.

In addition, the present invention also provides a host, a plant or microorganism transformed with the expression vector; the plant is selected from lettuce, spinach, tomato, radish, Chinese cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

The invention also provides a medicament which comprises the fusion protein and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a method for expressing the fusion protein of the Somallutamide and the lumbrokinase by taking the plant as a host, wherein the expression vector bombards the leaves by using a gene gun, a regeneration plant is obtained after the expression vector is expressed in a plant chlorophyll body, and the plant leaves are freeze-dried, crushed and filled into capsules to prepare the hypoglycemic capsule.

In some embodiments of the invention, the gene gun bombardment comprises the steps of:

step 1: preparing a vector for transformation;

step 2: preparing a particle bullet;

and step 3: bombardment with a gene gun;

and 4, step 4: and culturing and regenerating into plants after conversion.

The plant chloroplast expression technology is a technology for transferring plasmids containing target proteins into plant chloroplasts by using a gene gun bombardment and homologous recombination mode to obtain high-efficiency expression in the plant chloroplasts of the genes. Plant expression systems are very low cost, only one to two thousandths of them, compared to animal cell expression systems.

The invention fuses and expresses the somaglutide and lumbrokinase, and the oral administration is realized, thus relieving the pain of patients caused by long-term frequent injection.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a schematic diagram of the vector pUC 57-sem;

FIG. 2 shows the western-blot results; wherein lane 1 shows non-expressing plants; lane 2 shows the production of somaglutitade (Semaglutide) and lumbrokinase fusion protein from lettuce.

Detailed Description

The invention discloses application of plant production of oral dolaglutin and nattokinase hypoglycemic capsules, and technical personnel in the field can use the contents for reference and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention provides application of a plant as a host in expressing a fusion protein of Somatoxylum thamnaetin Semaglutide and lumbrokinase. Preferably, the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant. The invention also provides an expression vector, which comprises a fusion protein sequence of the Somatoxylum Semaglutide and lumbrokinase and a vector.

In some embodiments of the invention, the codon of the fusion protein of somaglutide and lumbrokinase is optimized to a plant-preferred codon.

In some embodiments of the invention, the optimized fusion protein sequence of the somaglutide and lumbrokinase is shown in SEQ ID No. 1; the nucleotide sequence of the optimized fusion protein of the Somatoxylum thatamide Semaglutide and the lumbrokinase is shown in SEQ ID No. 2.

In some embodiments of the invention, the vector is a plant chloroplast vector.

In some embodiments of the present invention, the method for constructing the expression vector comprises the following steps:

step 1: optimizing the codon of the fusion protein of the Somatoxylate Semaglutide and the lumbrokinase into a codon preferred by plants;

step 2: carrying out gene synthesis by using the Kisry and cloning into a pUC57 vector to obtain a pUC57-sem cloning vector;

specifically, in order to provide high-efficiency expression of the foreign protein in the plant, the invention obtains a nucleotide sequence from the amino acid sequence of the fusion protein of the Somatauutamide and lumbrokinase by utilizing reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss _ backsranseq /), optimizes the codon to the preferred codon of the plant, and synthesizes the codon by the Kinsley company (Nanjing, China). And cloned from Kinry into pUC57 vector to obtain pUC57-sem vector (FIG. 1).

The invention also provides application of the expression vector in expression of the fusion protein of the Somatoxylum Semaglutide and the lumbrokinase.

The expression vector provided by the invention bombards plant leaves with a gene gun, and the plant leaves are harvested after regeneration into plants and prepared into oral hypoglycemic capsules.

The invention carries out structural modification and modification on active polypeptide with the hypoglycemic effect to ensure that the active polypeptide can be absorbed by intestinal tracts and can reach the characteristic of effective treatment concentration in vivo, and plants are used for producing the active substance. Since lettuce is easy to grow and can be commercially mass-produced, it is more easily available and less expensive than other plants such as tobacco, etc., and since no complicated special production equipment is required, the cost can be significantly reduced. In conclusion, the invention can utilize the lettuce system to produce the fusion protein of the Somatoxylum thaumaglutide and the lumbrokinase in a large scale.

The invention utilizes plant leaves to produce oral hypoglycemic capsules. The product for reducing blood sugar does not need injection, relieves the pain of patients, and simultaneously, the product is a long-acting product for reducing blood sugar, and the patients can take the medicine once a week. The lettuce does not contain plant toxic substances, and the product does not need a protein purification process, thereby greatly shortening the production period and the production cost.

The raw materials and reagents used in the application of the plant provided by the invention as a host in expressing the fusion protein of the Somatoxylum thatamale Semaglutide and the lumbrokinase can be purchased from the market.

The invention is further illustrated by the following examples:

example 1 construction of chloroplast expression vectors

In order to efficiently express the foreign protein in the plant, a nucleotide sequence is obtained from the amino acid sequence of the fusion protein of the Semaglutide and the lumbrokinase by utilizing a reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss _ backstrans eq /), and the codon of the nucleotide sequence is optimized to a plant preferred codon, and the nucleotide sequence is synthesized by Kinsley corporation (Nanjing, China).

Example 2 conversion Material preparation

Soaking plant seeds in sterile water overnight, soaking in 70% ethanol for 1 min, and washing with sterile water for 1 time; then treating with 2% NaClO (with 0.1% Tween-20) for 15min, gently mixing for 1 time every 5min, and washing with sterile water for 4-5 times; the dried mixture is absorbed by sterile filter paper, planted on 1/2MS culture medium (containing 3% sucrose and 0.7% agar powder and having a pH value of 5.8), placed in a light incubator at 25 ℃, and cultured in the dark for 16h under light for 8h, and can be used for transformation in about 3 weeks.

Example 3 Gene gun preparation

50-60mg of gold powder (0.6 μm) was weighed into a dry 1.5mL sterile EP centrifuge tube. Add 1mL of absolute ethanol and vortex for 2 min. 1mL of sterile water was added, vortexed for 1 minute, allowed to stand at room temperature for 1 minute, centrifuged at 10,000rpm for 2 minutes, and the supernatant was removed. Add 1mL of 50% glycerol, resuspend the gold powder and freeze-preserve at-20 ℃.

The gold powder suspension in the glycerol storage state was vortexed for 5 minutes to resuspend the gold powder. Add 50. mu.L of the gold powder suspension to a sterile 1.5mL centrifuge tube and vortex for 1 min. Add 10. mu.g of plasmid DNA and vortex for 30 seconds. Add 50. mu.L of 2.5M CaCl2 and vortex for 30 seconds. mu.L of 0.1M spermidine was added and the mixture was vortexed for 5 minutes and allowed to stand on ice for 2 minutes. Add 60. mu.L of pre-cooled absolute ethanol, resuspend it by finger flick, centrifuge at 14,000rpm for 10 seconds, remove supernatant and repeat once. Add 50. mu.L of absolute ethanol to resuspend for use.

Example 4 particle gun bombardment

According to the number of samples, a certain number of carrier membranes, splittable membranes and blocking nets (note that the carrier membranes and the splittable membranes need to be replaced by each gun, and the same sample of the blocking net can be used together) are measured and soaked in absolute ethyl alcohol for 15 minutes, washed by sterile water for 2 times, and naturally dried for later use. And placing the dried carrier membrane into a sterile iron ring, and flattening. And (3) fully and uniformly mixing the prepared bullets in a vortex manner, putting 10 mu L of bullets in the center of a carrier film, and naturally drying. The particle emitting device was removed from the bombardment chamber, the lid was unscrewed, a blocking net was added, the particle slide was mounted in a fixed slot (the side with the particles facing downwards), the lid was screwed on, and the particle emitting device was returned to the bombardment chamber.

Example 5 post-transformation culture and screening

1. Dark culture: and (3) cutting down the bombarded lettuce leaves, and placing the leaves which are cut into 2 mm leaves and 10-20 mm leaves into an RMOL culture medium (without antibiotics) for dark culture at 25 ℃ for 2 days.

2. Screening and culturing: the dark culture-terminated material was transferred to a selection medium (antibiotic concentration 50. mu.g/mL) for selection culture.

3. Rooting culture: the shoots were transferred to rooting medium (antibiotic concentration 100. mu.g/mL) to induce rooting.

Example 6 Western blot detection of expression of target proteins

Grinding with liquid nitrogen, performing denaturing lysis to extract plant protein, mixing lysis supernatant and 5 × sample buffer (adding beta-mercaptoethanol to final concentration of 5% before use) at ratio of 4:1 (such as mixing 200 μ l protein lysis supernatant with 50 μ l 5 × sample buffer), mixing, heating at 95 deg.C for 6min, and treating negative control and positive control; the electrophoresis voltage is 80V, the separation gel is 120V, after the target protein is run to the middle position of the separation gel, the electrophoresis is stopped, the electrophoresis liquid in the lower tank is recovered, the electrophoresis device is disassembled, the negative electrode (black), sponge, filter paper, gel and PVDF membrane (which is activated by methanol for 15s and washed by ddH2O in advance and then soaked in 1 Xtransfer buffer solution) or NC membrane (which is not required to be activated), the filter paper, the sponge and the positive electrode (transparent) are placed in sequence, the assembly is carried out after air bubbles are removed, the electrophoresis tank is placed (the black side of the electrophoresis tank corresponding to the black side of the electrophoresis tank is injected), the transfer buffer solution is filled, the whole electrophoresis tank is placed in ice-water mixed liquid, and the 90V electrophoresis is carried out for 1.0 h; preparing 5% skimmed milk powder (sealing solution) at the end of electrophoresis, sealing the transferred membrane in the sealing solution at room temperature for at least 1h, and incubating at 4 deg.C for one time overnight (one time is diluted in 5% skimmed milk powder, and the dilution ratio is referred to the specification); washing the protein by PBST or TBST for 15min multiplied by 3 times, incubating a secondary antibody for 1-2 h at room temperature, washing the PBST or TBST for 15min multiplied by 3 times, developing by using a DAB kit, photographing, wherein the size of a gel display protein is 32kDa and is consistent with the molecular weight of an expected fusion protein of the Somatout Semaglutide and lumbrokinase, and the successful expression and purification of the target fusion protein are proved.

Example 7 detection of Activity of Semaglutide-lumbrokinase fusion protein

After a stabilization period lasting seven weeks, dogs were randomized into three treatment groups of 10 dogs each receiving one of two test capsules containing a hypoglycemic protein (the fusion protein of somatoditamide and lumbrokinase made in example 5 of the present invention) and no hypoglycemic protein, with commercially available somatoditamide (500 ng/g fed per body weight) as a positive control, and dogs were randomized into groups and received the same test diet to measure glycemic response after each repetition.

Dogs fasted 24 hours before blood glucose testing began. The catheterized site was shaved, aseptically treated, and catheterized into the right cephalic vein. Two baseline samples were taken approximately 5 minutes apart. Immediately after the last baseline sample was taken, the dogs were fed a diet equivalent to 1% of their body weight and contained 1 or 3 hypoglycemic capsules, which were allowed to eat for up to 15 minutes. If the dog did not eat the experimental diet within 15 minutes, his glycemic response was not detected the same day and was re-detected the following day. Additional blood samples were collected at 10, 20, 30, 45, 60, 120, 180 and 240 minutes after the meal. Blood samples were centrifuged at 1300 Xg for 15 minutes and two aliquots of 1ml plasma at each time point were cryopreserved within two hours after collection. Plasma glucose concentration (mg/dl) was determined using the hexokinase method. The test data collected are mean + -SD, and the positive control group is commercial Semaglutide.

TABLE 1 test results for sugar concentration in dog blood

Note: ^* shows significant difference (P is less than 0.05) compared with a control group without reducing the sugar; ^** comparison tool for comparison with control group without blood sugar reductionThe difference is very significant (P is less than 0.01);

the experimental result shows that compared with the commercial somaglutide, the fusion protein capsule containing the somaglutide and lumbrokinase can obviously reduce the content of dog blood sugar (P < 0.001). After 2 hours, the dog had a blood glucose concentration of 3.4. + -. 0.018mmol/L measured for oral administration of 3 fusion protein capsules, while the dog had a blood glucose concentration of 4.1. + -. 0.011mmol/L measured for 3 oral administrations of commercially available somaglutide. Indicating that the fusion protein is more resistant to decomposition by gastric acid than the commercial somaglutide, which is somaglutide, and thus readily enters the blood to interpret blood glucose.

Example 8 animal toxicity test

Experimental mice of 7 weeks size were randomly divided into three treatment groups of 10 mice each, and received a diet containing the hypoglycemic protein (500 ng/g fed by body weight) (the fusion protein of somaglutide and lumbrokinase obtained in the present invention), commercially available somaglutid (500 ng/g fed by body weight) as a positive control, and one of two experimental capsules without the hypoglycemic protein, and received the same experimental diet. The feeding is continuously carried out for 10 days, the observation is carried out after each feeding, the observation needs to be continuously carried out for more than 6 hours every day, the mice are not observed to be in an excited state or a suppressed state, phenomena such as slow action and the like do not occur, and diarrhea and the like do not occur. Proves that the oral administration safety of the fusion protein capsule of the somaglutide and the lumbrokinase is high.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> king league

Application of <120> plant in production of oral somaglutide and lumbrokinase fusion protein hypoglycemic capsules

<130> MP1902781

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 301

<212> PRT

<213> Somalutai (Fusion protein of Semaglutide 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> Somalutai (Fusion protein of Semaglutide 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

1. The application of the expression vector or the plant containing the expression vector in expressing the fusion protein of the thaumatin and the lumbrokinase or preparing the medicine containing the fusion protein; the plant is lettuce; the plant is selected from seeds, leaves, rhizomes or whole plants;

the medicine is an oral hypoglycemic preparation;

the construction method of the expression vector comprises the following steps:

step 1: optimizing the codon of the fusion protein of the thaumautput and the lumbrokinase into a codon preferred by plants, wherein the nucleotide sequence of the codon is shown as SEQ ID No. 2;

and 2, step: the nucleotide sequence was cloned into a pUC57 vector to obtain pUC 57-sem.