CN110218259B

CN110218259B - Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules

Info

Publication number: CN110218259B
Application number: CN201910550518.5A
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: 2022-09-16
Anticipated expiration: 2039-06-24
Also published as: WO2020259111A1; CN110218259A

Abstract

The invention relates to the field of biotechnology, in particular to application of glucagon-like peptide-1 short peptide and transferrin fusion protein produced by plants in preparing oral 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 results of biological activity tests show that the blood glucose reducing capsule produced by the platform technology obviously reduces the blood glucose concentration in dog blood.

Description

Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in preparing oral hypoglycemic capsules

Technical Field

The invention relates to the field of biotechnology, in particular to application of glucagon-like peptide-1 short peptide and transferrin fusion protein produced by plants in preparing oral hypoglycemic capsules.

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. The invention fuses and expresses transferrin and GLP-1, can realize oral administration, and relieves the pain of patients caused by long-term frequent injection.

Disclosure of Invention

In view of the above, the present invention provides an application of a fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in the manufacture of oral hypoglycemic capsules. The invention carries out structural modification and modification on the active polypeptide with the hypoglycemic effect, so that the active polypeptide has the characteristics of being absorbed by intestinal tracts and achieving effective treatment concentration in vivo, and the active substance is produced by plants. The invention uses plant, especially lettuce as the high-efficiency platform technology of recombinant protein production, and expresses the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin. And making into oral blood sugar lowering capsule.

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

the invention provides a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin, which has 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).

The invention provides nucleotides encoding said fusion proteins 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 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 shown in the (I), (II) or (III) and a nucleotide sequence which 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).

On the basis of the research, 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.

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

step 1: respectively optimizing codons of the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin to 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 pUC57 vector to obtain pGLP-1.

The invention also provides application of the expression vector or the plant in expressing the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin 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 drug is an oral formulation for lowering blood glucose.

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.

The invention also provides a method for expressing the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and the transferrin by taking the plant as a host, bombarding the leaf of the expression vector by using a gene gun, expressing the expression vector in the plant chlorophyll to obtain a regenerated plant, freeze-drying and crushing the plant leaf, and extracting to obtain the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and the transferrin.

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

step 1: preparing a vector for transformation;

step 2: preparing particle bullets;

and step 3: bombardment with a gene gun;

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

The invention also provides a method for preparing the hypoglycemic drug by taking the plant as a host, which comprises the steps of bombarding the leaf of the expression vector by using a gene gun, expressing the expression vector in a plant chlorophyll body to obtain a regenerated plant, freeze-drying, crushing and extracting the plant leaf to obtain the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and the transferrin, and filling.

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 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. Lettuce does not contain plant toxic substances, and the product does not need a protein purification process, so that the production period and the production cost can be greatly shortened.

Experiments show that the plant system, especially the lettuce system, is a more economic and efficient expression platform, and chloroplasts can efficiently express active protein. 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 a lettuce system to produce the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and the transferrin in a large scale.

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 representation of the vector pGLP-1;

FIG. 2 shows the results of a western-blot.

Detailed Description

The invention discloses an application of a fusion protein of glucagon-like peptide-1 short peptide produced by plants and transferrin in preparing oral hypoglycemic capsules, which can be realized by appropriately improving process parameters by referring to the content. 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 an application of plants in producing oral hypoglycemic capsules. The invention uses plant, especially lettuce as the high-efficiency platform technology of recombinant protein production, and expresses the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin. And making into oral blood sugar lowering capsule.

the invention provides application of a plant as a host in expressing fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin. 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 glucagon-like peptide-1 (GLP-1) short peptide and transferrin and a vector.

In some embodiments of the invention, the fusion protein codon of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin is optimized to a plant-preferred codon.

In some embodiments of the invention, the sequence of the fusion protein of the optimized glucagon-like peptide-1 (GLP-1) short peptide and transferrin is shown as SEQ ID No. 1; the nucleotide sequence of the fusion protein of the optimized glucagon-like peptide-1 (GLP-1) short peptide and transferrin 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 codons of a fusion protein of a glucagon-like peptide-1 (GLP-1) short peptide and transferrin to plant-preferred codons;

step 2: carrying out gene synthesis and cloning on the obtained product into a pUC57 vector by using the Kinry to obtain a pGLP-1 cloning vector;

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

The invention also provides application of the expression vector in expressing fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.

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 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 raw materials and reagents used in the application of the fusion protein of the plant-produced glucagon-like peptide-1 short peptide and transferrin to manufacture the oral hypoglycemic capsule are all available in 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 a fusion protein amino acid sequence of glucagon-like peptide-1 (GLP-1) short peptide and transferrin by utilizing reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss _ backsranseq /), and the codon of the nucleotide sequence is optimized to a codon preferred by the plant and synthesized by the Kinsley company (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 is removed from the bombardment chamber, the cover is screwed off, the blocking net is added, the particle slide is arranged in a fixed groove (the surface with the particles faces downwards), the cover is screwed on, and the particle emitting device is put back into 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 material after the completion of the dark culture was transferred to a selection medium (antibiotic concentration: 50. mu.g/mL) and subjected to selection culture.

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

Example 6Western blot detection of expression of target proteins

Grinding with liquid nitrogen, performing denaturing lysis to extract plant protein, mixing lysis supernatant and 5 × loading 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 l5 × loading 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 with PBST or TBST for 15min × 3 times, incubating with secondary antibody at room temperature for 1-2 h, washing with PBST or TBST for 15min × 3 times, developing with DAB kit, photographing, and analyzing the expression of target protein, as shown in FIG. 2: the result shows that the chloroplast transformation plant has a GLP-1 band, and the non-transformation plant has no expression band, which proves that GLP-1 is expressed in the lettuce leaves.

Example 7 detection of Activity of fusion protein of glucagon-like peptide-1 (GLP-1) short peptide with transferrin

After a stabilization period lasting seven weeks, dogs were randomized into two treatment groups of 3 dogs, each receiving one of two experimental capsules containing a hypoglycemic protein (the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide prepared in example 5 and transferrin) and no hypoglycemic protein, respectively, for the first repetition. The dogs were randomized again and received a different experimental diet for a second repeat. Replicates I and II were continued for at least 2 weeks and glycemic response was measured after the end of each replicate.

Dogs fasted 24 hours before blood glucose testing began. The catheterized site was shaved, aseptically treated, and the catheter was inserted 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.

TABLE 1 test results for sugar concentration in dog blood

Note: marked with significant difference (P < 0.05); with very significant differences (P < 0.01).

Example 8 animal toxicity test

Experimental mice of 7 weeks size were randomly divided into three treatment groups of 10 mice each, and received one of two experimental capsules containing a hypoglycemic protein (500 ng/g fed by body weight) (a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide obtained according to the present invention and transferrin) and no hypoglycemic protein, respectively, 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 of the glucagon-like peptide-1 (GLP-1) short peptide and the transferrin is high.

The comprehensive test results show that the plant system, especially the lettuce system, is a more economic and efficient expression platform. Can express recombinant protein rapidly and instantaneously, and can produce fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin in a short time and on a large scale.

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

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Application of fusion protein of glucagon-like peptide-1 short peptide and transferrin produced by plants in manufacturing oral hypoglycemic capsules

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gtacctgttc ttgctgaaaa ttatgaaaaa tctgataatt gtgaagatac tccagaagct 1680

ggatatttcg ctgttgctgt agttaaaaaa tctgcttctg atcttacttg ggataatctt 1740

aaggggaaaa aatcttgcca tactgctgta ggtagaactg ctggatggaa tattcctatg 1800

ggacttcttt ataataagat taatcattgt cgttttgatg aatttttctc tgaaggttgt 1860

gctcctggat ctaaaaaaga ttcttctctt tgtaaattat gtatgggatc tggtcttaat 1920

ctttgtgaac caaataacaa agaaggatat tatggttata ctggagcttt tagatgtctt 1980

gttgaaaaag gagatgttgc attcgttaaa catcaaactg tacctcaaaa tactggagga 2040

aaaaatcctg atccatgggc taaaaatctt aatgagaaag attatgaatt attatgttta 2100

gatggaacta gaaaacctgt agaagaatat gctaattgtc atcttgctag agctccaaat 2160

catgctgtag ttactcgtaa agataaagaa gcttgtgttc ataaaattct tcgtcaacaa 2220

caacatcttt ttggttctga agtaactgat tgttctggaa atttctgttt atttcgttct 2280

gaaactaaag atcttttatt tcgtgatgat actgtttgtt tagctaaact tcatgataga 2340

aatacttatg aaaaatatct tggtgaagaa tatgttaaag ctgttggaaa tcttcgtaaa 2400

tgttctactt cttctcttct tgaagcttgt acttttagac gtccttaa 2448

Claims

1. Use of an expression vector or a plant comprising said expression vector for expressing a fusion protein of a glucagon-like peptide-1 short peptide and transferrin or for the preparation of a medicament comprising said fusion protein; the plant is lettuce; the plant is selected from seeds, leaves, rhizomes or whole plants;

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

step 1: optimizing the codon of the fusion protein of the glucagon-like peptide-1 short peptide and transferrin into a codon 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 pUC57 vector to obtain pGLP-1.

2. The use of claim 1, wherein the medicament is an oral formulation for lowering blood glucose.

3. The medicine is characterized by comprising fusion protein coded by nucleic acid shown as SEQ ID No.2 and pharmaceutically acceptable auxiliary materials.

4. The medicament of claim 3, wherein the medicament is an oral formulation for lowering blood glucose.