WO2020259111A1 - 植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用 - Google Patents

植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用 Download PDF

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WO2020259111A1
WO2020259111A1 PCT/CN2020/089983 CN2020089983W WO2020259111A1 WO 2020259111 A1 WO2020259111 A1 WO 2020259111A1 CN 2020089983 W CN2020089983 W CN 2020089983W WO 2020259111 A1 WO2020259111 A1 WO 2020259111A1
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plant
peptide
nucleotide sequence
fusion protein
glucagon
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王跃驹
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王跃驹
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
    • C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the invention relates to the field of biotechnology, in particular to the application of plant production of a fusion protein of glucagon-like peptide-1 short peptide and transferrin to manufacture oral hypoglycemic capsules.
  • Diabetes is a common and frequently-occurring disease characterized by chronic hyperglycemia. It is a disorder of sugar, fat, and protein metabolism caused by defects in insulin secretion or action in the body, or both.
  • IDDM insulin-dependent
  • NIDDM non-insulin-dependent
  • Glucagon-like peptide-1 (Glucagon-like peptide-1, GLP-1) is an incretin secreted by intestinal endocrine cells. It is a post-translational processing product of proglucagon gene. There are many types in the body Existing form. It has the following physiological effects: it acts on pancreatic ⁇ cells in a glucose-dependent manner, promotes the transcription of insulin genes, increases insulin biosynthesis and secretion; stimulates the proliferation and differentiation of ⁇ cells, inhibits ⁇ cell apoptosis, thereby increasing the number of pancreatic ⁇ cells , Inhibit the secretion of glucagon, suppress appetite and ingestion, delay the emptying of stomach contents, etc. These functions are conducive to reducing blood glucose after meals and maintaining a constant level of blood glucose.
  • natural GLP-1 has many advantages in the treatment of diabetes, its half-life in vivo is only about 2 minutes, which limits its direct clinical application. Mutation of certain amino acids in natural GLP-1 can prolong its half-life under the condition of ensuring its activity, and it can maintain normal blood glucose levels by once a week.
  • Related products currently on the market include Liraglutide, Dulaglutide, Semaglutide, etc. Due to the nature of peptide drugs and the various barriers created by the human body, injection has always been the main route of their conventional administration.
  • the present invention expresses transferrin and GLP-1 in fusion, can realize oral administration, and alleviate the pain caused by long-term frequent injection of patients.
  • the present invention provides an application of plant production of a fusion protein of glucagon-like peptide-1 short peptide and transferrin to manufacture oral hypoglycemic capsules.
  • the present invention carries out structural transformation and modification on the active polypeptide with hypoglycemic effect, so that it can be absorbed through the intestinal tract and reach an effective therapeutic concentration in the body, and the active substance is produced by plants.
  • the invention uses plants, especially lettuce, as an efficient platform technology for recombinant protein production, and expresses a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin. And make oral hypoglycemic capsules.
  • GLP-1 glucagon-like peptide-1
  • the present invention provides a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin, which has:
  • the present invention provides nucleotides encoding the fusion protein, having
  • (III) A nucleotide sequence that encodes the same protein as the nucleotide sequence of (I) or (II), but is different from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or
  • nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (I), (II) or (III), and with (I), (II) or (III) nucleotide sequences with the same or similar functions; or
  • (V) a nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (I), (II), (III) or (IV).
  • the present invention also provides an expression vector, including the nucleotide and the vector to be transformed.
  • the vector to be transformed is a chloroplast expression vector.
  • the present invention also provides a method for constructing the expression vector, which includes the following steps:
  • Step 1 The codons of the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin are optimized to plant-preferred codons, and the nucleotide sequence is as SEQ ID No. 2 shown;
  • Step 2 Cloning the nucleotide sequence into the pUC57 vector to obtain pGLP-1.
  • the present invention also provides the application of the expression vector or plant in expressing the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin or preparing a medicine containing the fusion protein;
  • the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the organ of the plant is selected from seed, leaf, rhizome or whole plant.
  • the drug is a hypoglycemic oral preparation.
  • 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, cabbage, corn, soybean, wheat or tobacco; the organ of the plant is selected from Seeds, leaves, rhizomes or whole plants.
  • the invention also provides medicines, including the fusion protein and pharmaceutically acceptable excipients.
  • the drug is a hypoglycemic oral preparation.
  • the present invention also provides a method for expressing the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin in a plant as a host.
  • the expression vector is used to bombard the leaves with a gene gun. Regenerated plants are obtained after expression in chloroplasts, and plant leaves are freeze-dried, crushed, and extracted to obtain a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.
  • the gene gun bombardment includes the following steps:
  • Step 1 Prepare vector for transformation
  • Step 2 Prepare particle bullets
  • Step 3 Gene gun bombardment
  • Step 4 After conversion, cultivate and regenerate into plants.
  • the present invention also provides a method for preparing a hypoglycemic drug by using a plant as a host.
  • the expression vector is bombarded with a gene gun to the leaves and expressed in plant chloroplasts to obtain regenerated plants.
  • the plant leaves are freeze-dried, crushed, and extracted to obtain Glucagon-like peptide-1 (GLP-1) short peptide and transferrin fusion protein, filling.
  • GLP-1 Glucagon-like peptide-1
  • the gene gun bombardment includes the following steps:
  • Step 1 Prepare vector for transformation
  • Step 2 Prepare particle bullets
  • Step 3 Gene gun bombardment
  • Step 4 After conversion, cultivate and regenerate into plants.
  • the invention uses plant leaves to produce oral hypoglycemic capsules.
  • the hypoglycemic product does not require injections, which reduces the pain of patients.
  • this product is a long-acting hypoglycemic product that patients can take once a week. Lettuce does not contain plant toxic substances, and this product does not require a protein purification process, which can greatly shorten the production cycle and production costs.
  • the present invention found through experiments that the plant system, especially the lettuce system, is a more economical and efficient expression platform, and the chloroplast can efficiently express active proteins. Because lettuce is easy to grow and can be produced in large quantities commercially, it is easier to obtain and cheaper than other plants, such as tobacco, and because it does not require complex special production equipment, the cost can be significantly reduced. In summary, the present invention can utilize the lettuce system to produce a large-scale fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.
  • GLP-1 glucagon-like peptide-1
  • Figure 1 shows a schematic diagram of the vector pGLP-1
  • FIG. 1 shows the western-blot results.
  • the invention discloses the application of a plant to produce a fusion protein of glucagon-like peptide-1 short peptide and transferrin to manufacture oral hypoglycemic capsules.
  • the invention provides the application of plant production of oral hypoglycemic capsules.
  • the invention uses plants, especially lettuce, as an efficient platform technology for recombinant protein production, and expresses a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin. And make oral hypoglycemic capsules.
  • GLP-1 glucagon-like peptide-1
  • the invention provides the application of plants as a host in expressing a fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.
  • the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; and the organ of the plant is selected from seeds, leaves, rhizomes or whole plants.
  • the present invention also provides an expression vector, including the fusion protein sequence of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin and the vector.
  • the codon of the fusion protein of the glucagon-like peptide-1 (GLP-1) short peptide and transferrin is optimized to a plant-preferred codon.
  • the sequence of the fusion protein of the optimized glucagon-like peptide-1 (GLP-1) short peptide and transferrin is shown in SEQ ID No. 1; the optimized The nucleotide sequence of the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin is shown in SEQ ID No.2.
  • the vector is a plant chloroplast vector.
  • the construction method of the expression vector includes the following steps:
  • Step 1 Optimize the codons of the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin to plant-preferred codons;
  • Step 2 Gene synthesis and cloning into pUC57 vector by GenScript to obtain pGLP-1 cloning vector
  • the present invention uses the amino acid sequence of the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin using reverse translation software (https:/ /www.ebi.ac.uk/Tools/st/emboss_backtranseq/) to obtain the nucleotide sequence, and optimize its codons to plant-preferred codons, synthesized by GenScript (Nanjing, China). And cloned into the pUC57 vector from GenScript to obtain the pGLP-1 vector ( Figure 1).
  • reverse translation software https:/ /www.ebi.ac.uk/Tools/st/emboss_backtranseq/
  • the invention also provides the application of the expression vector in expressing the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.
  • GLP-1 glucagon-like peptide-1
  • the expression vector provided by the present invention is bombarded with gene gun to plant leaves, regenerated into plants, and then harvested and made into oral hypoglycemic capsules.
  • Plant chloroplast expression technology is the use of gene gun bombardment and homologous recombination to transfer a plasmid containing the target protein to plant chloroplasts to obtain high-efficiency expression of the gene in plant chloroplasts. Compared with animal cell expression systems, the cost of plant expression systems is very low, only one thousandth to two thousandths.
  • the invention uses plant leaves to produce oral hypoglycemic capsules.
  • the hypoglycemic product does not require injections, which reduces the pain of patients.
  • this product is a long-acting hypoglycemic product that patients can take once a week. Lettuce does not contain plant toxic substances, and this product does not require a protein purification process, which can greatly shorten the production cycle and production costs.
  • the present invention found through experiments that the plant system, especially the lettuce system, is a more economical and efficient expression platform, and the chloroplast can efficiently express active proteins. Because lettuce is easy to grow and can be produced in large quantities commercially, it is easier to obtain and cheaper than other plants, such as tobacco, and because it does not require complex special production equipment, the cost can be significantly reduced. In summary, the present invention can utilize the lettuce system to produce a large-scale fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin.
  • GLP-1 glucagon-like peptide-1
  • the raw materials and reagents used in the application of the plant for producing the fusion protein of glucagon-like peptide-1 short peptide and transferrin to produce oral hypoglycemic capsules can be purchased from the market.
  • GLP-1 glucagon-like peptide-1
  • transferrin reverse translation software (https://www.ebi. ac.uk/Tools/st/emboss_backtranseq/) to obtain the nucleotide sequence and optimize its codons to plant-preferred codons, synthesized by GenScript (Nanjing, China).
  • the gold powder suspension in the glycerol state was vortexed for 5 minutes to resuspend the gold powder. Take 50 ⁇ L of gold powder suspension in a sterile 1.5mL centrifuge tube and vortex for 1 minute. Add 10 ⁇ g plasmid DNA and vortex for 30 seconds. Add 50 ⁇ L 2.5M CaCl2 and vortex for 30 seconds. Add 20 ⁇ L 0.1M spermidine, vortex the mixture for 5 minutes, and let stand on ice for 2 minutes. Add 60 ⁇ L of pre-cooled absolute ethanol, flick your fingers to resuspend, centrifuge at 14,000 rpm for 10 seconds, remove the supernatant, and repeat. Add 50 ⁇ L of absolute ethanol to resuspend and set aside.
  • carrier membranes and splittable membranes, and barrier nets are used according to the number of samples (note: carrier membranes and splittable membranes need to be replaced every gun, and the barrier net can be shared with the same sample) soak in absolute ethanol for 15 minutes, and use sterile Rinse twice with water, let it dry naturally, and set aside. Put the dried carrier film into a sterile iron ring and flatten it. The prepared bullets were vortexed to mix well, and 10 ⁇ L bullets were placed in the center of the carrier film and dried naturally. Move the particle launcher out of the bombardment chamber, unscrew the cover, add the blocking net, install the particle slide in the fixed groove (the side with the particles is facing down), screw on the cover, and put the particle launcher back into the bombardment chamber.
  • Screening culture transfer the materials after dark culture to the screening medium (antibiotic concentration of 50 ⁇ g/mL) for screening culture.
  • Rooting culture transfer the buds to a rooting medium (antibiotic concentration of 100 ⁇ g/mL) to induce rooting.
  • the dogs were randomly divided into two treatment groups, 3 in each group, and received the glucagon-like peptide-1 (GLP-1) prepared in Example 5 A fusion protein of short peptide and transferrin) and one of the two experimental capsules without hypoglycemic protein. Repeat for the first time. Randomly group the dogs again, accept a different experimental diet, and do a second repetition. Repeat I and II for at least 2 weeks, and check the blood glucose response after each repetition.
  • GLP-1 glucagon-like peptide-1
  • the dog fasts for 24 hours. Shave the hair at the catheter insertion site, aseptically process, and insert the catheter into the right cephalic vein. Take two baseline samples approximately 5 minutes apart. After the last baseline sample was collected, the dog was immediately fed a diet equivalent to 1% of its body weight and containing 1 or 3 hypoglycemic capsules, and allowed to eat for up to 15 minutes. If the dog does not eat the experimental diet within 15 minutes, the blood glucose response will not be tested on the same day, and the test will be repeated the next day. At 10, 20, 30, 45, 60, 120, 180, and 240 minutes after eating, additional blood samples were collected.
  • the blood samples were centrifuged at 1300 ⁇ g for 15 minutes, and two aliquots of 1ml plasma at each time point were cryopreserved within two hours after collection.
  • the hexokinase method was used to determine the plasma glucose concentration (mg/dl).
  • the 7-week-old experimental mice were randomly divided into three treatment groups, each with 10 mice, and received glycoprotein-containing (fed 500ng/g according to body weight) (glucagon-like peptide-1 ( GLP-1) short peptide and transferrin fusion protein), and one of the two experimental capsules without hypoglycemic protein, received the same experimental diet. Feeding was continued for 10 days, and observations were made after each feeding. Continuous observation was required for more than 6 hours a day. It did not see whether the mice were excited or inhibited, did not appear to be slow or diarrhea. It proves that the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin has high oral safety.
  • the above test results show that the plant system, especially the lettuce system, is a more economical and efficient expression platform.
  • the recombinant protein can be expressed quickly and transiently, and the fusion protein of glucagon-like peptide-1 (GLP-1) short peptide and transferrin can be produced on a large scale in a short time.
  • GLP-1 glucagon-like peptide-1

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Abstract

本发明属于生物技术领域，提供了利用植物生产的胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白以及该蛋白制造口服降糖胶囊的应用。本发明利用植物如生菜作为重组蛋白质生产的表达平台，表达体系简单、高效，将生产该重组蛋白质的叶片冻干制成胶囊，该胶囊可以在常温保存并保持生物活性，能显著降低狗血液中的血糖浓度。

Description

植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用
本申请要求于2019年06月24日提交中国专利局、申请号为201910550518.5、发明名称为“植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，特别涉及植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用。
背景技术
糖尿病是以慢性高血糖为特征的常见病、多发病，是由体内胰岛素分泌或作用缺陷，或二者同时存在而引起的糖、脂肪、蛋白质代谢紊乱。临床上主要有胰岛素依赖型(IDDM，I型)和非胰岛素依赖型(NIDDM，II型)两种类型。随着生活水平提高，无论在发达国家还是在发展中国家，糖尿病的发病率都在逐年上升。糖尿病作为一种严重的非传染性慢性疾病现已成为全世界各国密切关注的重大公共卫生问题之一，是全球范围内继心血管和肿瘤疾病之后的第三号杀手。从世界卫生组织公布的数据来看，1995年全球糖尿病患者仅3000万人左右，而到了1997年已增至1.35亿，到2030年将有3亿II型糖尿病患者，患者增幅最快的地区为亚洲和非洲。II型糖尿病患者的传统治疗模式一般是遵循饮食控制、口服抗糖尿病药物和外源性胰岛素的阶梯式治疗。但目前糖尿病治疗领域仍存在着诸多尚待解决的重要问题，而且还存在一些副作用和限制。
胰高血糖素样肽-1(Glucagon-like peptide-1,GLP-1)是由肠道内分泌细胞分泌的肠降血糖素，是胰高血糖素原基因翻译后加工产物，在体内有多种存在形式。它具有以下生理作用：以葡萄糖依赖方式作用于胰岛β细胞，促进胰岛素基因的转录，增加胰岛素的生物合成和分泌；刺激β细胞的增殖和分化，抑制β细胞凋亡，从而增加胰岛β细胞数量，抑制胰高血 糖素的分泌，抑制食欲及摄食，延缓胃内容物排空等。这些功能都有利于降低餐后血糖并使血糖维持在恒定水平。
尽管天然GLP-1在治疗糖尿病上有诸多优点，但它的体内半衰期仅为2分钟左右，限制了其在临床上的直接应用。而将天然的GLP-1中的某些氨基酸突变后可以在保证其活性的条件下延长它的半衰期，做到每周一次给药就可以保持正常的血糖水平。目前已经上市的相关产品有Liraglutide、Dulaglutide、Semaglutide等。由于多肽类药物本身的性质以及人体对其产生的各种屏障，其常规给药途经一直以注射为主。本发明将转铁蛋白与GLP-1融合表达，可以实现在口服给药，减轻病患长期频繁注射带来的痛苦。
发明内容
有鉴于此，本发明提供一种植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用。本发明对具有降糖作用的活性多肽进行结构改造和修饰，使其获得可通过肠道进行吸收并在体内达到有效治疗浓度的特性，并通过植物来生产该活性物质。本发明利用植物尤其是生菜作为重组蛋白生产的高效平台技术，表达了胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。并且制成口服降糖胶囊。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白，其具有：
(Ⅰ)、如SEQ ID No.1所示的氨基酸序列；或
(Ⅱ)、如(Ⅰ)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(Ⅰ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
(III)、与(Ⅰ)或(Ⅱ)所述序列至少有80％同源性的氨基酸序列。
本发明提供了编码所述融合蛋白的核苷酸，具有
(Ⅰ)、如SEQ ID No.2所示的核苷酸序列；或
(Ⅱ)、如SEQ ID 2所示的核苷酸序列的互补核苷酸序列；或
(Ⅲ)、与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的核苷酸序列；或
(Ⅳ)、与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列功能相同或相似的核苷酸序列；或
(V)、与(Ⅰ)、(Ⅱ)、(Ⅲ)或(Ⅳ)所述核苷酸序列至少有80％同源性的核苷酸序列。
在上述的研究基础上，本发明还提供了表达载体，包括所述的核苷酸以及待转化载体。
在本发明的一些具体实施方案中，所述待转化载体为叶绿体表达载体。
本发明还提供了所述的表达载体的构建方法，包括如下步骤：
步骤1：分别将所述胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白的密码子优化为植物偏好的密码子，其核苷酸序列如SEQ ID No.2所示；
步骤2：将所述核苷酸序列克隆到pUC57载体中，获得pGLP-1。
本发明还提供了所述的表达载体或植物在表达胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白或制备包含所述融合蛋白的药物中的应用；所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草；所述植物的器官选自种子、叶、根茎或整株植物。
在本发明的一些具体实施方案中，所述药物为降糖的口服制剂。
此外，本发明还提供了宿主，转化有所述表达载体的植物或微生物；所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草；所述植物的器官选自种子、叶、根茎或整株植物。
本发明还提供了药物，包括所述的融合蛋白以及药学上可接受的辅料。
在本发明的一些具体实施方案中，所述药物为降糖的口服制剂。
本发明还提供了一种植物作为宿主表达胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白的方法，将所述的表达载体用基因枪轰击叶 片，在植物叶绿体中表达后获得再生植株，将植物叶片冻干粉碎、提取，获得胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。
在本发明的一些具体实施方案中，所述基因枪轰击包括如下步骤：
步骤1：准备转化用载体；
步骤2：准备微粒子弹；
步骤3：基因枪轰击；
步骤4：转换后培养、再生为植株。
本发明还提供了一种植物作为宿主制备降糖的药物的方法，将所述的表达载体用基因枪轰击叶片，在植物叶绿体中表达后获得再生植株，将植物叶片冻干粉碎、提取，获得胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白，灌装。
在本发明的一些具体实施方案中，所述基因枪轰击包括如下步骤：
步骤1：准备转化用载体；
步骤2：准备微粒子弹；
步骤3：基因枪轰击；
步骤4：转换后培养、再生为植株。
本发明利用植物叶片，生产口服降糖胶囊。该降糖产品不需要注射，减轻病患的痛苦，同时这一产品为长效降糖产品，病患可以做到一周用药一次。生菜不含有植物有毒物质，而且本产品不需蛋白纯化流程，可以大大缩短生产周期和生产成本。
本发明通过实验发现，植物系统尤其是生菜系统是更加经济、高效的表达平台，叶绿体可以高效的表达活性蛋白。由于生菜易于生长并且可商业上大量生产，因此比其它植物，如烟草等更容易获得并且更便宜，并且由于不需要复杂的特殊生产设备，成本可显著降低。综上所述，本发明可以利用生菜系统大规模生产胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对 实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示载体pGLP-1示意图；
图2示western-blot结果。
具体实施方式
本发明公开了一种植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供了植物生产口服降糖胶囊的应用。本发明利用植物尤其是生菜作为重组蛋白生产的高效平台技术，表达了胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。并且制成口服降糖胶囊。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了植物作为宿主在表达胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白的应用。优选的，所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草；所述植物的器官选自种子、叶、根茎或整株植物。本发明还提供了一种表达载体，包括胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白序列以及载体。
在本发明的一些具体实施方案中，所述胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白密码子优化为植物偏好的密码子。
在本发明的一些具体实施方案中，所述优化的胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白序列如SEQ ID No.1所示；所述优化的胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白的核苷酸序列如SEQ ID No.2所示。
在本发明的一些具体实施方案中，所述载体为植物叶绿体载体。
在本发明的一些具体实施方案中，所述表达载体的构建方法包括如下 步骤：
步骤1：将胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白的密码子优化为植物偏好的密码子；
步骤2：由金斯瑞进行基因合成克隆到pUC57载体中，获得pGLP-1克隆载体；
具体的，为了提供外源蛋白在植物中的高效表达，本发明将胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白氨基酸序列利用反翻译软件(https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/)得到核苷酸序列，并将其密码子优化为植物偏好的密码子，由金斯瑞公司(南京，中国)合成。并由金斯瑞克隆到pUC57载体中，获得pGLP-1载体(图1)。
本发明还提供了所述的表达载体在表达胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白中的应用。
将本发明提供的表达载体用基因枪轰击植物叶片，再生为植株后收获植物叶片并制成口服降糖胶囊。
植物叶绿体表达技术是利用基因枪轰击、同源重组的方式将含有目标蛋白的质粒转移到植物叶绿体中，获得该基因植物叶绿体中高效表达的技术。与动物细胞表达系统相比，植物表达系统的成本非常低，仅为其千分之一到千分之二。
本发明利用植物叶片，生产口服降糖胶囊。该降糖产品不需要注射，减轻病患的痛苦，同时这一产品为长效降糖产品，病患可以做到一周用药一次。生菜不含有植物有毒物质，而且本产品不需蛋白纯化流程，可以大大缩短生产周期和生产成本。
本发明通过实验发现，植物系统尤其是生菜系统是更加经济、高效的表达平台，叶绿体可以高效的表达活性蛋白。由于生菜易于生长并且可商业上大量生产，因此比其它植物，如烟草等更容易获得并且更便宜，并且由于不需要复杂的特殊生产设备，成本可显著降低。综上所述，本发明可以利用生菜系统大规模生产胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。
本发明提供的植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋 白制造口服降糖胶囊的应用中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1 叶绿体表达载体的构建
为了将外源蛋白在植物中的高效表达，将胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白氨基酸序列利用反翻译软件(https://www.ebi.ac.uk/Tools/st/emboss_backtranseq/)得到核苷酸序列，并将其密码子优化为植物偏好的密码子，由金斯瑞公司(南京，中国)合成。
实施例2 转化材料准备
将植物种子用无菌水浸泡过夜，用70％乙醇浸泡1分钟后用无菌水冲洗1次；再用2％NaClO(加0.1％Tween-20)处理15分钟，每5分钟轻柔混匀1次，无菌水冲洗4-5次；用无菌滤纸吸干后种植于1/2MS培养基上(含3％蔗糖、0.7％琼脂粉、pH值为5.8)，置于光照培养箱中25℃，16h光照8h黑暗培养，约3周可用于转化。
实施例3 基因枪准备
称取50-60mg金粉(0.6μm)于干燥的1.5mL灭菌EP离心管。加入1mL无水乙醇，涡旋2分钟。加入1mL无菌水，涡旋1分钟，室温放置1分钟，10,000rpm离心2分钟，去上清。加入1mL 50％甘油，重悬金粉，-20℃冻存。
甘油保存状态的金粉悬液涡旋5分钟使金粉重悬。取50μL金粉悬液于无菌1.5mL离心管，涡旋1分钟。加入10μg质粒DNA，涡旋30秒。加入50μL 2.5M CaCl2，涡旋30秒。加入20μL 0.1M亚精胺，混合物涡旋5分钟，冰上静置2分钟。加60μL预冷的无水乙醇，手指轻弹使之重悬，14,000rpm离心10秒，去上清，重复一次。加入50μL无水乙醇重悬，备用。
实施例4 基因枪轰击
根据样品数量取一定数量的载体膜、可裂膜、阻挡网(注意：载体膜、可裂膜需每枪更换，阻挡网同一个样品可共用)于无水乙醇中浸泡15分钟，用无菌水冲洗2次，自然晾干，备用。将晾干的载体膜放入无菌铁环中，压平。将制备好的子弹涡旋充分混匀，取10μL子弹于载体膜中央，自然晾干。把微粒发射装置移出轰击室，旋下盖子，加入阻挡网，把微粒载片安装在固定槽中(有微粒的一面朝下)，旋上盖子，将微粒发射装置放回轰击室。
实施例5 转化后培养及筛选
1.暗培养：将轰击后的生菜叶片剪下，切成10～20mm2的叶盘置于RMOL培养基(不加抗生素)中25℃暗培养2天。
2.筛选培养：将暗培养结束的材料转移至筛选培养基(抗生素浓度为50μg/mL)中进行筛选培养。
3.生根培养：将芽转移至生根培养基(抗生素浓度为100μg/mL)中诱导生根。
实施例6 Western blot检测目的蛋白表达情况
采用液氮研磨、变性裂解提取植物蛋白，将裂解上清和5×上样缓冲液(使用前加入β-巯基乙醇至终浓度为5％)按4:1的比例混合(如200μl蛋白裂解上清与50μl5×上样缓冲液混合)，混匀，95℃加热6min，同时处理阴性对照及阳性对照；电泳电压积层胶80V，分离胶120V，待目的蛋白跑至分离胶中间位置后，停止电泳，回收下槽电泳液，拆开电泳装置，按照负极(黑色)、海绵、滤纸、凝胶、PVDF膜(事先用甲醇活化15s、ddH2O洗涤后浸泡于1×转移缓冲液中)或NC膜(不需活化)、滤纸、海绵、正极(透明)的顺序放置，排气泡后组装，放入电泳槽(注黑色对应电泳槽黑色一面放入)，加满转移缓冲液，将整个电泳槽放入冰水混合液中，90V电泳1.0h；电泳快结束时配制5％脱脂奶粉(封闭液)，将转移后的膜放入封闭液中室温封闭至少1h，4℃孵育一抗过夜(一抗稀释于 5％脱脂奶粉中，稀释比参考说明书)；使用PBST或TBST洗涤15min×3次，室温孵育二抗1～2h，PBST或TBST洗涤15min×3次，采用DAB试剂盒进行显色，拍照，分析目的蛋白表达情况，如图2所示：结果表明叶绿体转化植株有GLP-1条带，非转化植株没有表达条带，证明GLP-1已在生菜叶片中表达。
实施例7 胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白活性检测
在持续七周的稳定期后，将狗随机分为两个治疗组，每组3只，分别接收含有降糖蛋白(实施例5制得的胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白)及不含降糖蛋白两种实验胶囊中的一种，做第一次重复。再次将狗随机分组，接受另一种不同的实验饮食，做第二次重复。重复I和II至少持续2周，在每次重复结束后检测血糖反应。
在血糖检测开始前，狗禁食24小时。剃去导管插入部位的毛，无菌处理，导管插入右头静脉。大约间隔5分钟采集两基线样品。在采集最后一个基线样品后，立即给狗喂相当其体重1％的饮食并含有1片或3片降糖胶囊，至多允许其吃15分钟。如果在15分钟内狗不吃实验饮食，则当天不检测其血糖反应，次日重新检测。在进食后10、20、30、45、60、120、180和240分钟，采集额外的血液样品。血液样品1300×g离心15分钟，在采集后两小时内将每个时间点1ml血浆之两等分样品冻存。应用己糖激酶方法测定血浆葡萄糖浓度(mg/dl)。
表1 狗血液中糖浓度的实验结果
Figure PCTCN2020089983-appb-000001
Figure PCTCN2020089983-appb-000002
注：*示具有显著差异(P＜0.05)；**示具有极显著差异(P＜0.01)。
实施例8 动物毒性试验
将7周大小的实验用小白鼠随机分为三个治疗组，每组10只，分别接收含有降糖蛋白(按照体重喂食500ng/g)(本发明得到的胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白)，及不含降糖蛋白两种实验胶囊中的一种，接受相同的实验饮食。连续喂食10天，每次喂食后实进行观察，每天需要连续观察6小时以上，并没有看小鼠处于兴奋状态还是抑制状态，没有出现行动迟缓等现象，也没有出现腹泻等情况。证明胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白口服安全性高。
综合上述试验结果表明，植物系统尤其是生菜系统是更加经济、高效的表达平台。能够快速瞬时表达重组蛋白质，可以在短时间内大规模生产胰高血糖素样肽-1(GLP-1)短肽与转铁蛋白的融合蛋白。
以上对本发明所提供的植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (10)

  1. 胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白，其特征在于，其具有：
    (Ⅰ)、如SEQ ID No.1所示的氨基酸序列；或
    (Ⅱ)、如(Ⅰ)所述的氨基酸序列经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列，且与(Ⅰ)所示的氨基酸序列功能相同或相似的氨基酸序列；或
    (III)、与(Ⅰ)或(Ⅱ)所述序列至少有80％同源性的氨基酸序列。
  2. 编码如权利要求1所述融合蛋白的核苷酸，其特征在于，具有
    (Ⅰ)、如SEQ ID No.2所示的核苷酸序列；或
    (Ⅱ)、如SEQ ID 2所示的核苷酸序列的互补核苷酸序列；或
    (Ⅲ)、与(Ⅰ)或(Ⅱ)的核苷酸序列编码相同蛋白质，但因遗传密码的简并性而与(Ⅰ)或(Ⅱ)的核苷酸序列不同的核苷酸序列；或
    (Ⅳ)、与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列经取代、缺失或添加一个或多个核苷酸序列获得的核苷酸序列，且与(Ⅰ)、(Ⅱ)或(Ⅲ)所示的核苷酸序列功能相同或相似的核苷酸序列；或
    (V)、与(Ⅰ)、(Ⅱ)、(Ⅲ)或(Ⅳ)所述核苷酸序列至少有80％同源性的核苷酸序列。
  3. 表达载体，其特征在于，包括如权利要求2所述的核苷酸以及待转化载体。
  4. 如权利要求3所述的表达载体，其特征在于，所述待转化载体为叶绿体表达载体。
  5. 如权利要求3或4所述的表达载体的构建方法，其特征在于，包括如下步骤：
    步骤1：分别将所述胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白的密码子优化为植物偏好的密码子，其核苷酸序列如SEQ ID No.2所示；
    步骤2：将所述核苷酸序列克隆到pUC57载体中，获得pGLP-1。
  6. 如权利要求3或4所述的表达载体或植物在表达胰高血糖素样肽 -1短肽与转铁蛋白的融合蛋白或制备包含所述融合蛋白的药物中的应用；所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草；所述植物的器官选自种子、叶、根茎或整株植物。
  7. 如权利要求6所述的应用，其特征在于，所述药物为降糖的口服制剂。
  8. 宿主，其特征在于，转化有如权利要求3或4所述表达载体的植物或微生物；所述植物选自生菜、菠菜、番茄、萝卜、白菜、玉米、大豆、小麦或烟草；所述植物的器官选自种子、叶、根茎或整株植物。
  9. 药物，其特征在于，包括如权利要求1所述的融合蛋白以及药学上可接受的辅料。
  10. 如权利要求9所述的药物，其特征在于，所述药物为降糖的口服制剂。
PCT/CN2020/089983 2019-06-24 2020-05-13 植物生产胰高血糖素样肽-1短肽与转铁蛋白的融合蛋白制造口服降糖胶囊的应用 WO2020259111A1 (zh)

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