WO2019036859A1 - 分泌glp-1及其衍生物的蛋白表达系统及其制备方法和用途 - Google Patents

分泌glp-1及其衍生物的蛋白表达系统及其制备方法和用途 Download PDF

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WO2019036859A1
WO2019036859A1 PCT/CN2017/098344 CN2017098344W WO2019036859A1 WO 2019036859 A1 WO2019036859 A1 WO 2019036859A1 CN 2017098344 W CN2017098344 W CN 2017098344W WO 2019036859 A1 WO2019036859 A1 WO 2019036859A1
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glp
mglp
lactobacillus
expression vector
nucleotide sequence
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金万洙
董孟
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中国科学院动物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • C—CHEMISTRY; METALLURGY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
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    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/23—Lactobacillus acidophilus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/245—Lactobacillus casei
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention belongs to the field of biopharmaceuticals, and in particular, the present invention relates to a protein expression system for expressing GLP-1 and its derivatives, a preparation method thereof and use thereof.
  • Glucagon-like peptide-1 (GLP-1) is a peptide hormone encoded by the human glucagon gene and secreted by intestinal L cells. It has the following physiological effects: glucose-dependent Acts on islet ⁇ cells, promotes insulin gene transcription, increases insulin biosynthesis and secretion; stimulates ⁇ cell proliferation and differentiation, inhibits ⁇ cell apoptosis, thereby increasing islet ⁇ cell number, inhibiting glucagon secretion, inhibiting Appetite and food intake, delaying the emptying of stomach contents. These features help to reduce postprandial blood glucose and maintain blood sugar at a constant level.
  • GLP-1 glucagon-like peptide-1
  • GIP glucose-dependent insulinotropic polypeptide
  • GLP-1 has a stronger insulin-promoting effect than GIP, and has a physiological effect of inhibiting postprandial glucagon secretion, delaying gastric emptying, and inhibiting GIP, and thus, GLP-1. More suitable than GIP to become a target for the treatment of T2DM.
  • GLP-1 is glucose-dependent, does not cause hypoglycemia when treating diabetes, and avoids the risk of hypoglycemia that is often present in the treatment of diabetes. Therefore, it is an ideal candidate for the treatment of type 2 diabetes.
  • oral administration is a major drawback.
  • Patentes CN101824388A and CN1865430 disclose methods for expressing and producing GLP-1 using Pichia pastoris;
  • patents CN102660491A and CN1587385 disclose methods for expressing and producing GLP-1 using E. coli;
  • Patent CN1861635 discloses the use of E. coli for expression and production of GLP-2 Methods. The use of yeast E.
  • GLP-1 and GLP-2 are produced by genetic engineering. 2 must be purified and injected for use, not directly for oral administration.
  • the nisin controlled expression system is an ideal food-grade induced expression system for controlling protein production, and the expression of heterologous protein by lactic acid bacteria is superior to cytoplasmic expression.
  • the lactic acid bacteria food-grade secretory expression vector PNZ8149 has a large defect: pNZ8149 can only express foreign proteins in the cytoplasm, but does not secrete the function of the expressed foreign protein. Lactic acid bacteria have thicker cell walls and express proteins in the cytoplasm, which is not conducive to protein production.
  • the object of the present invention is to solve the problem that the current use of GLP-1 can not be taken orally, to provide a kind of colonization in the intestine, and to continuously secrete GLP-1, which has obvious therapeutic effects on diabetic patients and obese patients.
  • the recombinant intestinal normal bacteria the present invention also provides the preparation method and use of the normal intestinal bacteria.
  • the present inventors have found that by using genetic engineering technology, transforming the expression vector pNZ8149, and inserting the signal peptide spUSP45, a novel expression vector having a function of secreting and expressing a foreign protein in a normal intestinal bacterium can be constructed. Further, the enhancer sequence LEISS is added to the vector to enhance its secretory expression ability. Further, the synthetic human GLP-1 sequence was cloned into the expression vector pNZ8149 and transformed into a host strain, and the food-grade extracellular secretion-inducing expression system of the present application can be obtained. The system is able to colonize the gut and continuously secrete GLP-1, for diabetics and fat Fat patients have obvious therapeutic effects and are expected to produce considerable social and economic benefits.
  • the invention provides a GLP-1 analog mGLP-1, the sequence of the mGLP-1 is set forth in SEQ ID NO:4;
  • the present invention also provides a nucleotide sequence encoding the mGLP-1;
  • nucleotide sequence is as shown in SEQ ID NO: 2.
  • the present invention provides a recombinant expression vector for expressing GLP-1, which comprises a signal peptide, an enhancer sequence, an enterokinase recognition site, a nucleotide sequence of human GLP-1, or a nucleotide sequence of the expression vector pNZ8149 or a nucleotide sequence of human mGLP-1 or a related derivative thereof;
  • the signal peptide is spUSP45; more preferably, the sequence of the signal peptide is as shown in SEQ ID NO: 5;
  • the enhancer sequence is LEISS;
  • the enterokinase recognition site is DDDDK.
  • the enterokinase recognition site is recognized and cleaved by enterokinase present in the human duodenal mucosa, thereby producing a protein identical to the in vivo source sequence;
  • the signal peptide is inserted between the multiple cloning sites NcoI and SacI of the expression vector pNZ8149;
  • nucleotide sequence of the human GLP-1 is as shown in SEQ ID NO: 1;
  • nucleotide sequence of the human mGLP-1 is as shown in SEQ ID NO: 2;
  • the derivative is a nucleotide sequence encoding a related mutant, analog and agonist of said GLP-1 or mGLP-1.
  • the present invention provides a protein expression system for secreting GLP-1, which is a normal intestinal bacterium transformed with the above recombinant expression vector.
  • the normal intestinal bacteria is selected from the group consisting of Lactobacillus, Bifidobacterium or Lactobacillus;
  • the lactic acid bacteria refers to all strains of the genus Lactobacillus contained in the "List of Species for Foods" issued by the Ministry of Health (Wei Jian Zhi Fa [2010] No. 65), namely: Lactobacillus acidophilus (Lactobacillus) Acidophilus), Lactobacillus casei, Lactobacillus crispatus, Lactobacillus bulgaricus, Lactobacillus delbrueckii, Lactobacillus fermentium, Lactobacillus gasseri ), Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus salivarius, or Lactococcus lactis subspecies lactis;
  • the lactic acid bacterium is L. 1 actis NZ3900.
  • the protein expression system secreting GLP-1 is lactic acid bacteria NICE (the nisin) Controlled expression) system.
  • the inventors of the present invention found that the expression vector pNZ8149 was inserted into the enterokinase recognition site (DDDDK) by using genetic engineering technology, and the synthetic human GLP nucleotide sequence was cloned into the expression vector pNZ8149 and transformed into the host strain.
  • the host bacteria can produce GLP-1 and/or mGLP-1 or a derivative thereof in the intestinal tract under the action of enterokinase.
  • the protein expression system of the present application can effectively function under the induction of nisin (Nisin) at a concentration of (0.1-25 ng/ml).
  • the present invention provides a method of producing a GLP-1 secreting protein expression system of the present invention, comprising the steps of:
  • the synthetic sequence NcoI-SPusp45-LEISS-DDDDK-GLP-1-SacI or NcoI-SPusp45-LEISS-DDDDK-mGLP-1-SacI;
  • step 2) inserting the sequence synthesized in step 1) into the expression vector pNZ8149 to construct a recombinant expression vector expressing GLP-1;
  • step 2) transforming the recombinant expression vector of step 2) into a normal intestinal bacterium
  • the recombinant intestinal normal bacteria obtained in the step 3) are inoculated in a medium to culture and express.
  • the present invention provides the use of the GLP-1 analog mGLP-1 of the present invention, a recombinant expression vector expressing GLP-1 or a protein expression system secreting GLP-1 in the preparation of a medicament;
  • the medicament is for preventing and/or treating diabetes, obesity, diabetic kidney injury, diabetic macroangiopathy, diabetic microvascular injury, diabetic retinopathy, glucagonemia, necrotic migratory erythema, glucagon Metabolic diseases such as tumors, coronary heart disease, hypertensive heart disease, valvular heart disease, alcoholic cardiomyopathy, cardiovascular complications of diabetes, and drugs for treating nerve damage diseases such as Alzheimer's and Parkinson's.
  • diabetes obesity, diabetic kidney injury, diabetic macroangiopathy, diabetic microvascular injury, diabetic retinopathy, glucagonemia, necrotic migratory erythema, glucagon Metabolic diseases such as tumors, coronary heart disease, hypertensive heart disease, valvular heart disease, alcoholic cardiomyopathy, cardiovascular complications of diabetes, and drugs for treating nerve damage diseases such as Alzheimer's and Parkinson's.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a GLP-1 analog mGLP-1, a recombinant expression vector expressing GLP-1 or a protein expression secreting GLP-1 system;
  • the pharmaceutical composition is an oral pharmaceutical composition.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and/or adjuvant.
  • the present invention has the following advantages:
  • the clinically used GLP-1 administration method is injection.
  • the patient needs a special environment, such as the supervision and operation of the hospital and specialized medical personnel to complete the treatment.
  • the present invention provides an oral type of GLP-1, which greatly improves the patient's medication compliance.
  • the GLP-1 protein expression system L.1actis NZ3900-GLP1, NZ3900-mGLP-1 provided by the present invention can be used for the treatment of diabetes and related metabolic syndrome.
  • Figure 1 is an electrophoresis pattern of PCR verification of the GLP-1 protein expression vector PNZ8149-GLP-1 of the present invention.
  • Figure 2 is an electrophoresis pattern of PCR verification of the GLP-1 protein expression vector PNZ8149-mGLP-1 of the present invention.
  • Fig. 3 is a liquid chromatogram of protein expression of the NZ3900-GLP-1 strain of the present invention.
  • Figure 4 is a liquid chromatogram of protein expression of the NZ3900-mGLP-1 strain of the present invention.
  • Fig. 5 is a diagram showing the results of sequencing of protein expression of the NZ3900-GLP-1 strain of the present invention.
  • Fig. 6 is a diagram showing the results of sequencing of protein expression of the NZ3900-mGLP-1 strain of the present invention.
  • Fig. 7 is a diagram showing the in vitro DPP-IV digestion degradation of GLP-1 and mGLP-1 proteins of the present invention.
  • Figure 8 is a graph showing the therapeutic effects of the NZ3900-GLP-1 and NZ3900-mGLP-1 strains of the present invention on a Db/Db type 2 diabetic mouse model.
  • the reagents used in the following examples are all analytical grade reagents and are commercially available from regular sources.
  • the GLP-1 protein expression vectors pNZ8149-GLP-1 and pNZ8149-mGLP-1 of the present invention were constructed using the edible grade protein expression vector pNZ8149 (MoBiTec, Germany), respectively.
  • signal peptide spUSP45 is inserted into the multiple cloning site NcoI of the expression vector pNZ8149 Between SacI and SacI.
  • the enterokinase recognition site DDDDK
  • the GLP-1 protein expression vectors pNZ8149-GLP-1 and pNZ8149-mGLP-1 of the present application can be cleaved in the intestinal tract under the action of enterokinase, and produced in the human body.
  • the same structure of the source GLP-1 as well as mGLP-1 ensures its correct function.
  • GLP-1 and/or mGLP-1 were inserted between the protein expression vector pNZ8149 restriction enzyme site NcoI and the SacI restriction enzyme site.
  • the above DNA was synthesized by Shanghai Shenggong Bioengineering Co., Ltd.
  • GLP-1 GLP-1
  • mGLP-1 produced by the GLP-1 protein expression vectors PNZ8149-GLP-1 and PNZ8149-mGLP-1 constructed using the method of the present invention have no other modifications or other protein tags and are not antigenic.
  • mGLP-1 was mutated at the second amino acid of GLP-1.
  • the coding nucleotide sequence of GLP-1 is shown in SEQ ID NO: 1:
  • the coding nucleotide sequence of mGLP-1 is set forth in SEQ ID NO: 2:
  • amino acid sequence of GLP-1 is shown in SEQ ID NO: 3:
  • amino acid sequence of mGLP-1 is shown in SEQ ID NO: 4:
  • sequence of the signal peptide spUSP45 is shown in SEQ ID NO: 5:
  • the coding nucleotide sequence of the enhancer sequence LEISS is shown in SEQ ID NO:6:
  • the nucleotide sequence encoding the enterokinase recognition site DDDDK is shown in SEQ ID NO:7:
  • the above-described synthetic GLP-1 protein expression vectors pNZ8149-GLP-1 and pNZ8149-mGLP-1 were electroporated.
  • the method was transformed into competent cells of L.lactis NZ3900 strain, and the positive transforming bacteria were screened by Elliker selection medium, and the selected transforming bacteria were cultured, and the plasmid was extracted from the cells, and PCR was carried out (Fig. 1-2), sequencing identification, and identification results.
  • the strain is a protein strain secreting GLP-1, mGLP-1 secreted by the constructed NZ3900 L. Iactis, and named as NZ3900-GLP-1, NZ3900-mGLP-1.
  • Plasmid pNZ8149, pUC57-GLP-1 was extracted using the Tiangen Plasmid Mini Kit;
  • C was prepared by 1% agarose gel electrophoresis, electrophoresis at 120V for 30min; recovery at 234bp and 2500bp;
  • a 200 ⁇ l of the bacterial solution in a L. lactis NZ3900 strain storage tube stored at -80 ° C was inoculated into 5 ml of liquid GSGM17 medium, placed in a CO 2 incubator, and allowed to stand overnight at 30 ° C, 5% CO 2 .
  • the cells were resuspended in ice-cold 200 ml Wash II (0.5 mol/L sucrose, 100 ml/L glycerol, 50 mM EDTA), allowed to stand on ice for 15 min, centrifuged at 4000 r/min and centrifuged at 4 ° C for 20 min, and the cells were collected;
  • the cells were suspended in 4 ml of ice-cold lotion I (0.5 M sucrose, 100 ml/L glycerol), and placed in a 0.5 ml EP tube in a prior ice bath, 40 ⁇ l per tube, and stored at -80 °C.
  • ice-cold lotion I 0.5 M sucrose, 100 ml/L glycerol
  • the NZ3900-GLP-1 and NZ3900-mGLP-1 strains were cultured in M17 broth at 30 degrees (weigh 21.125 g of M17 broth medium, dissolved in 400 ml of distilled water, and adjusted to pH 7.2 with 10 mol/L NaOH. The volume was adjusted to 500ml, autoclaved, and the medium used was purchased from Beijing Aoboxing Biotechnology Co., Ltd.). When the OD value reached 0.4, the expression of GLP-1 and mGLP-1 protein was induced by 25 ng/ml Nisin. . Since GLP-1 is a polypeptide fragment of 31 amino acids in length, we performed HPLC-MASS analysis on the secreted supernatant after induction.
  • the strain was inserted into M17 liquid medium, and cultured at 30 ° C overnight, transferred to a new M17 medium at a dose of 1-2%, cultured to an OD600 nm of 0.4, and an appropriate amount of nisin nisin solution was added.
  • the final concentration of nisin was 25 ng/mL, and incubation was continued for 5 h at 30 °C. After the end of the culture, centrifugation was performed at 10,000 rpm for 5 min, and the supernatant of the medium was used for HPLC-MASS detection.
  • mGLP-1 is resistant to DPP-IV digestion and prolong the half-life
  • mGLP-1 polypeptides the above amino acid sequence was synthesized by Nanjing Kingsray Bioengineering Co., Ltd.
  • the above GLP-1 and mGLP-1 proteins were subjected to in vitro enzymatic hydrolysis experiments using purified human DPP-IV protease (ProSpec, #enz-375).
  • the results showed that the stability of mGLP-1 was significantly increased as shown in Fig. 8, and the stability was increased by 50% with respect to GLP-1. It is indicated that the mGLP-1 provided by the invention can significantly resist the degradation of DPP-IV and has greater potential for treatment in vivo. effect.
  • Example 5 NZ3900-GLP-1 strain, NZ3900-mGLP-1 strain for treatment of Db/Db mice Test
  • mice Eight-week-old, male B6-Db/Db obese mice (purchased from the Model Animal Research Institute of Nanjing University) were selected as experimental subjects, and were divided into three groups according to body weight and blood glucose before the experiment, with 8 rats in each group. Mice were given free diet for a period of time (fed 10 Kcal% low fat diet, Research diet, D12450B).
  • Oral GLP-1, mGLP-1 group oral dose of 1X10 12 NZ3900-GLP-1, NZ3900-mGLP-1 strain / day / time
  • control group or oral dose of 1X10 12 NZ3900 empty vector strain /day/time.
  • Figure 9 shows the effects of oral GLP-1 and mGLP-1 on glucose tolerance levels in mice.

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Abstract

提供了一种氨基酸序列如SEQ ID NO:4所示的GLP-1类似物mGLP-1，以及一种可在食品级乳酸菌中分泌表达GLP-1的重组表达载体和一种分泌GLP-1的蛋白表达系统，所述载体是在表达载体pNZ8149中插入信号肽spUSP45、增强子序列LEISS、肠激酶识别位点、人源GLP-1核酸序列或人源mGLP-1的核酸序列构建而成，所述表达系统为转化了上述表达载体的肠道正常菌。还提供了所述GLP-1类似物、其核酸序列、所述表达载体和表达系统在制备药物中的用途。

Description

分泌GLP-1及其衍生物的蛋白表达系统及其制备方法和用途 技术领域
本发明属于生物制药领域，具体而言，本发明涉及表达GLP-1及其衍生物的蛋白表达系统及其制备方法和用途。
背景技术
胰高血糖素样肽-1(glucagon-likepeptide1，GLP-1)是由人胰高血糖素基因编码，并由肠道L细胞分泌的一种肽类激素，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。
早在上世纪60年代，麦金太尔(Mcintyre)和埃尔里克(Elrick)等人就发现，葡萄糖口服可比静脉输注引起更强的胰岛素分泌，提示存在能够促进胰岛素分泌的内源性物质，该物质仅在口服葡萄糖时分泌，呈葡萄糖依赖式地增加胰岛素的释放。后来人们发现，小肠内分泌细胞产生的一种激素可调理胰岛对进餐的反响，该激素在餐后会促进胰岛素的过量分泌，因此被命名为肠促胰素(Incretin)，餐后胰岛素总量的60％是在其作用下分泌的。进一步研究显示，人体的肠促胰素主要有两种，分别是胰高血糖素样肽-1(GLP-1)和葡萄糖依赖的促胰岛素多肽(GIP)，其作用均由相应的特异性受体介导。人体研讨显示，2型糖尿病(T2DM)患者口服和输注葡萄糖后，胰岛素分泌量的差别降低，提示肠促胰素作用削弱；T2DM患者餐后GIP程度与健康人相当，而GLP-1的程度明显低于健康人，标明T2DM患者存在GLP-1缺乏。另一方面，GLP-1较GIP具有更强的促胰岛素分泌作用，并有抑制餐后胰高血糖素分泌、延缓胃排空、抑制摄食等GIP所不具有的生理作用，因而，GLP-1比GIP更合适成为医治T2DM的靶点。
然而，天然GLP-1的血浆半衰期仅为1-2分钟，极易被体内的二肽基肽酶Ⅳ(DPP-Ⅳ)降解，代谢不稳定性限制其作为药物的应用。为解决这一难题，当前有两种解决方法，一种是开发GLP-1类似物，让其既具有GLP-1的活性和功效，又能抵抗降解二肽基肽酶Ⅳ，例如现有的专利技术中，CN00806548.9、CN99814187.9、CN200410017667.9均为针对人源GLP-1序列的酶降解关键位点，进行结构改造，从而提高其代谢稳定性。但是这些改 造只考虑了酶降解一个消除因素，达不到理想的效果；另一种是开发DPP-Ⅳ抑制剂，使体内自身分泌的GLP-1不被降解，例如已上市的药物西格列汀(sitagliptin)、维格列汀(vildagliptin)、沙格列汀(saxagliptin)、阿格列汀(alogliptin)、利格列汀(linagliptin)、吉格列汀(gemigliptin)和替格列汀(teneligliptin)等。
但是，GLP-1是葡萄糖依赖性的，治疗糖尿病时不会出现低血糖症状，避免了糖尿病治疗中常存在的产生低血糖症的危险，因此是一种十分理想的治疗II型糖尿病的候选药物。在实际应用中，由于GLP-1是多肽，不能口服给药是其一大缺憾。专利CN101824388A和CN1865430公布了使用毕赤酵母表达和生产GLP-1的方法；专利CN102660491A和CN1587385公布了使用大肠杆菌表达和生产GLP-1的方法；专利CN1861635公布了使用大肠杆菌表达和生产GLP-2的方法。使用酵母的大肠杆菌，通过基因工程手段表达和生产GLP-1和GLP-2是一种经济、高效的方法，但由于酵母和大肠杆菌都不是食品级微生物，所生产的GLP-1和GLP-2必须经过纯化后注射使用，不能直接用于口服。
乳酸菌NICE(the nisin controlled expression)系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统，而乳酸菌分泌表达异源蛋白比细胞质表达更优越。但作为蛋白表达载体，乳酸菌食品级分泌表达载体PNZ8149存在很大缺陷：pNZ8149只能将外源蛋白表达于细胞质中，但是没有分泌表达的外源蛋白的功能。而乳酸菌具有较厚的细胞壁，将蛋白表达于胞质中，不利于蛋白产生作用。
因此，当前对能直接口服并作用于肠道的GLP-1的重组肠道正常菌存在需求。
发明内容
基于以上不足，本发明的目的是为解决当前使用GLP-1治疗时，无法口服的问题，提供一种能够定植于肠道内，持续分泌GLP-1，对糖尿病患者和肥胖患者具有明显的治疗作用的重组肠道正常菌，本发明还提供了该肠道正常菌的制备方法和用途。
本发明人发现，应用基因工程技术，改造表达载体pNZ8149，并插入信号肽spUSP45，可以构建一种新型的表达载体，其具有在肠道正常菌中分泌表达外源蛋白的功能。进一步地，将增强子序列LEISS加入该载体中，增强其分泌表达能力。更进一步地，把合成的人GLP-1序列克隆到该表达载体pNZ8149上，并转化到宿主菌中，可以获得本申请的食品级细胞外分泌诱导表达系统。该系统能够定植于肠道内，持续分泌GLP-1，对糖尿病患者和肥 胖患者具有明显的治疗作用，有望产生可观的社会和经济效益。
一方面，本发明提供了一种GLP-1类似物mGLP-1，所述mGLP-1的序列如SEQ ID NO:4所示；
本发明还提供了编码所述mGLP-1的核苷酸序列；
优选地，所述核苷酸序列如SEQ ID NO:2所示。
另一方面，本发明还提供了一种表达GLP-1的重组表达载体，其在表达载体pNZ8149插入信号肽、增强子序列、肠激酶识别位点、人源GLP-1的核苷酸序列或人源mGLP-1的核苷酸序列或其相关衍生物；
优选地，所述信号肽为spUSP45；更优选地，所述信号肽的序列如SEQ ID NO:5所示；
优选地，所述增强子序列为LEISS；
优选地，所述肠激酶识别位点为DDDDK。
所述肠激酶识别位点被存在于人十二指肠粘膜中的肠激酶识别并切割，进而产生与机体内源序列相同的蛋白；
优选地，所述信号肽插入到表达载体pNZ8149的多克隆位点NcoI和SacI之间；
优选地，所述人源GLP-1的核苷酸序列如SEQ ID NO:1所示；
优选地，所述人源mGLP-1的核苷酸序列如SEQ ID NO:2所示；
优选地，所述衍生物为编码所述GLP-1或mGLP-1的相关突变体，类似物以及激动剂的核苷酸序列。
另一方面，本发明提供了一种分泌GLP-1的蛋白表达系统，所述表达系统为被上述重组表达载体转化的肠道正常菌。
优选地，所述肠道正常菌选自乳酸杆菌、双歧杆菌或乳酸菌；
更优选地，所述乳酸菌为指卫生部颁发的《可用于食品的菌种名单》(卫办监督发〔2010〕65号)所包含的所有乳酸菌属所有菌株，即：嗜酸乳杆菌(Lactobacillus acidophilus)、干酪乳杆菌(Lactobacillus casei)、卷曲乳杆菌(Lactobacillus crispatus)、保加利亚乳杆菌(Lactobacillusbulgaricus)、德氏乳杆菌(Lactobacillus delbrueckii)、发酵乳杆菌(Lactobacillus fermentium)、格氏乳杆菌(Lactobacillus gasseri)、瑞士乳杆菌(Lactobacillus helveticus)、约氏乳杆菌(Lactobacillus johnsonii)、副干酪乳杆菌(Lactobacillus paracasei)、植物乳杆菌(Lactobacillusplantarum)、罗伊氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、唾液乳杆菌(Lactobacillus salivarius)，或乳酸乳球菌乳酸亚种(Lactococcuslactis subspecies lactis)等；
更优选地，所述乳酸菌为L.1actis NZ3900。
优选地，所述分泌GLP-1的蛋白表达系统为乳酸菌NICE(the nisin  controlled expression)系统。
本发明的发明人发现，应用基因工程技术，改造表达载体pNZ8149，插入肠激酶识别位点(DDDDK)，把合成的人GLP核苷酸序列克隆到该表达载体pNZ8149上，并转化到宿主菌中，该宿主菌可以在肠道内在肠激酶的作用下产生GLP-1和/或mGLP-1或其衍生物。并且，本申请的蛋白表达系统是在一定(0.1-25ng/ml)浓度的乳酸链球菌素(Nisin)诱导下才能有效的发挥功能。
再一方面，本发明提供了本发明的分泌GLP-1的蛋白表达系统的制备方法，其包括以下步骤：
1)人工合成GLP-1类似物mGLP-1、GLP-1或编码所述GLP-1或mGLP-1的相关突变体，类似物以及激动剂的核苷酸序列，上下游酶切位点分别为NcoI和SacI；
优选地，人工合成序列NcoI-SPusp45-LEISS-DDDDK-GLP-1-SacI或NcoI-SPusp45-LEISS-DDDDK-mGLP-1-SacI；
2)将步骤1)合成的序列插入表达载体pNZ8149中，构建表达GLP-1的重组表达载体；
3)将步骤2)的重组表达载体转化至肠道正常菌中；
4)将步骤3)得到的重组肠道正常菌接种于培养基中培养表达。
再另一方面，本发明提供了本发明的GLP-1类似物mGLP-1、表达GLP-1的重组表达载体或分泌GLP-1的蛋白表达系统在制备药物中的用途；
优选地，所述药物为预防和/治疗糖尿病、肥胖、糖尿病肾损伤、糖尿病大血管病变、糖尿病微血管损伤、糖尿病性视网膜病变、胰高血糖素血症、坏死性游走性红斑、高血糖素瘤等代谢性疾病、冠心病、高血压性心脏病、瓣膜性心脏病、酒精性心肌病、糖尿病心血管并发症以及用于治疗阿尔兹海默和帕金森等神经损伤性疾病的药物。
再另一方面，本发明提供了一种药物组合物，所述药物组合物包括治疗有效量的GLP-1类似物mGLP-1、表达GLP-1的重组表达载体或分泌GLP-1的蛋白表达系统；
优选地，所述药物组合物为口服药物组合物。
优选地，所述药物组合物包括药学上可用的载体和/或辅料。
与现有技术相比，本发明具有以下优点：
1)现在临床上使用的GLP-1给药方式是注射，病人需要特殊的环境，譬如医院和专门的医护人员的监督指导和操作下才能完成治疗。本发明提供一种口服型的GLP-1，极大地提高了患者的服药依从性。
2)从本申请提供的具体实施方案可以得知，在体外DPP-IV酶切实验中，mGLP-1的稳定性显著增加，表明本申请的突变体mGLP-1可以显著抵抗DPP-IV的体外酶解失活作用，显著增加了GLP-1的半衰期。
3)通过Db/Db糖尿病小鼠模型实验得知，本发明提供的mGLP-1表达菌株可以有效的改善糖尿病小鼠的高血糖症状。
综上所述，本发明提供的GLP-1蛋白表达系统L.1actis NZ3900-GLP1、NZ3900-mGLP-1可以用于糖尿病以及相关代谢综合征的治疗。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，在附图中，*表示p<0.05，**表示p<0.01，***表示p<0.001：
图1为本发明的GLP-1蛋白表达载体PNZ8149-GLP-1的PCR验证的电泳图。
图2为本发明的GLP-1蛋白表达载体PNZ8149-mGLP-1的PCR验证的电泳图。
图3为本发明的NZ3900-GLP-1菌株的蛋白表达液相色谱图。
图4为本发明的NZ3900-mGLP-1菌株的蛋白表达液相色谱图。
图5为本发明的NZ3900-GLP-1菌株的蛋白表达测序结果图。
图6为本发明的NZ3900-mGLP-1菌株的蛋白表达测序结果图。
图7为本发明的GLP-1、mGLP-1蛋白体外DPP-IV酶切降解实验图。
图8为本发明的NZ3900-GLP-1、NZ3900-mGLP-1菌株在Db/Db二型糖尿病小鼠模型上的治疗效果图。
实施发明的最佳方式
下面结合附图和具体实施例来详细说明本发明。应理解，以下实施例仅用于说明本发明而不用于限制本发明的范围。
以下实施例中所用的试剂均为分析纯级别的试剂，且可从正规渠道商购获得。
实施例1：GLP-1蛋白表达载体的设计与构建
利用食用级别的蛋白表达载体pNZ8149(MoBiTec，Germany)分别构建本发明的GLP-1蛋白表达载体pNZ8149-GLP-1和pNZ8149-mGLP-1。
其中，所述信号肽spUSP45插入到表达载体pNZ8149的多克隆位点NcoI 和SacI之间。
所述肠激酶识别位点(DDDDK)，因此，本申请GLP-1蛋白表达载体pNZ8149-GLP-1和pNZ8149-mGLP-1可以在肠道内在肠激酶的作用下被剪切，产生与人体内源性GLP-1相同的结构以及mGLP-1，确保其正确的功能发挥。
在蛋白表达载体pNZ8149限制性酶切位点NcoI和SacI限制性酶切位点之间插入了GLP-1和/或mGLP-1。
上述DNA由上海生工生物工程有限公司合成。
使用本发明的方法构建的GLP-1蛋白表达载体PNZ8149-GLP-1和PNZ8149-mGLP-1生产出来的GLP-1、mGLP-1没有其他修饰或其他蛋白标签，不具有抗原性。其中mGLP-1是在GLP-1第二位氨基酸处进行了突变.
GLP-1的编码核苷酸序列如SEQ ID NO:1所示：
Figure PCTCN2017098344-appb-000001
mGLP-1的编码核苷酸序列如SEQ ID NO:2所示：
Figure PCTCN2017098344-appb-000002
GLP-1的氨基酸序列如SEQ ID NO:3所示：
Figure PCTCN2017098344-appb-000003
mGLP-1的氨基酸序列如SEQ ID NO:4所示：
SEQ ID NO:4:
Figure PCTCN2017098344-appb-000004
信号肽spUSP45的序列如SEQ ID NO:5所示:
SEQ ID NO:5
Figure PCTCN2017098344-appb-000005
增强子序列LEISS的编码核苷酸序列如SEQ ID NO:6所示:
SEQ ID NO:6
Figure PCTCN2017098344-appb-000006
肠激酶识别位点DDDDK的编码核苷酸序列如SEQ ID NO:7所示：
SEQ ID NO:7
Figure PCTCN2017098344-appb-000007
实施例2：GLP-1、mGLP-1蛋白表达载体的验证
为了验证GLP-1蛋白表达载体pNZ8149-GLP-1和pNZ8149-mGLP-1构建的成功与否，把上述合成好的GLP-1蛋白表达载体pNZ8149-GLP-1和pNZ8149-mGLP-1用电穿孔法转化到L.lactis NZ3900菌株感受态细胞，并采用Elliker选择培养基筛选阳性转化菌，培养筛选出的转化菌，从菌体提取质粒，进行PCR(图1-2)、测序鉴定，鉴定结果为核苷酸序列与预期相符，该菌株即为所构建的NZ3900 L.Iactis分泌表达GLP-1、mGLP-1的蛋白菌株，将其命名为：NZ3900-GLP-1、NZ3900-mGLP-1。
具体方法如下：
一、pNZ8149-GLP-1重组质粒构建
1、NcoI-SPusp45-LEISS-DDDDK-GLP-1-SacI全序列化学合成
获得pUC57-GLP-1重组质粒；
2、构建pNZ8149-GLP-1重组质粒
A 采用天根质粒小提试剂盒，提取pNZ8149，pUC57-GLP-1质粒；
B 酶切
pNZ8149，pUC57-GLP-1酶切体系如下表1所示，总体系为50μL，37℃酶切3h；
表1酶切体系
Figure PCTCN2017098344-appb-000008
C 配制1％琼脂糖凝胶电泳，120V电压下电泳30min；在234bp和2500bp切胶回收；
D 酶连
酶连体系如下，总体系20μl，插入片段：载体＝7：1，16℃过夜；
表2酶连体系
Figure PCTCN2017098344-appb-000009
E 连接产物纯化回收
制备好的连接产物中加入2.5倍体积的无水乙醇，1/10体积的2.5mol/L乙酸钠，混匀后于-20℃放置lh，12000r/min离心5min，弃上清；用1ml 75％乙醇沉淀一次，12000r/min离心5min，弃上清，室温干燥20min，用去离子水溶解沉淀。
二、pNZ8149-GLP-1电转乳酸乳球菌NZ3900
1、乳酸乳球菌NZ3900感受态制备
A 从-80℃贮存的L.lactis NZ3900菌种保存管中取200μl菌液接种于5ml液体GSGM17培养基中，放入CO2培养箱，30℃、5％CO2静置培养过夜。
B 取5ml菌液加入50ml GSGM17培养基中，30℃、5％CO2静置培养12h。
C 取50ml菌液转种于400ml GSGM17培养基中，30℃、5％CO2静置培养至OD600约为0.3。
D 将菌液4000r/min、4℃离心20min，收集菌液；
E 用冰冷的400ml的洗液I(0.5mol/L蔗糖，100ml/L甘油)洗涤一次，4000r/min、4℃离心20min，收集菌液；
F 用冰冷的200ml洗液II(0.5mol/L蔗糖，100ml/L甘油，50mM EDTA)重悬菌体，冰上静置15min；4000r/min、4℃离心20min，收集菌体；
G 再用冰冷的洗液I(0.5M蔗糖，100ml/L甘油)100ml重悬菌体，4000r/min、4℃离心20min，收集菌体；
H 用4ml冰冷的洗液I(0.5M蔗糖，100ml/L甘油)悬浮菌体，分装入事先冰浴的0.5ml EP管中，每管40μl，储存于-80℃。
2、乳酸乳球菌NZ3900电转化
A 吸取40μL新鲜制备的感受态细菌悬液于冰冷的无菌EP管中，冰浴5min。
B 吸取1μl纯化后的连接产物加入感受态细菌，混匀后，置于冰上5min。
C 将细菌悬液和质粒DNA混合液加入冰冷的电转杯中，放入电击仪，设置条件为2500V，200Ω，25μF；
D 电击完毕后，迅速加入1ml冰冷的GM17-MC恢复培养基；混匀后转入EP管中冰浴5～10min；30℃培养2h。
E 取100μl恢复培养的菌液接种于Elliker选择培养基，30℃过夜培养，挑取黄色菌落进行鉴定。
实施例3：GLP-1、mGLP-1蛋白的诱导与表达
把NZ3900-GLP-1、NZ3900-mGLP-1菌株在30度的M17肉汤培养基(称取21.125g M17肉汤培养基，溶于400ml蒸馏水中，用10mol/L NaOH调pH值至7.2，定容至500ml，高压灭菌，所用培养基购于北京奥博星生物技术有限责任公司)中培养，等OD值达到0.4的时候用25ng/ml的Nisin诱导GLP-1、mGLP-1蛋白的表达。鉴于GLP-1是长度为31个氨基酸的多肽片段，我们对诱导后的分泌上清液进行了HPLC-MASS分析。如图3、4的HPLC结果所示，在Nisin(25ng/ml)的处理情况下GLP-1、mGLP-1被特异性诱导表达。之后进行了质谱分析，如图5、6所示，Nisin诱导表达的蛋白是GLP-1、mGLP-1蛋白。
具体方法如下：
HPLC-MASS检测pNZ8149-GLP-1在乳酸乳球菌中的分泌表达
1、pNZ8149-GLP-1在乳酸乳球菌中的诱导分泌表达
将菌株接入M17液体培养基中，30℃静置培养过夜，按1-2％接种量转入新的M17培养基中，培养至OD600 nm为0.4，加入适量乳链菌素nisin使用液，使得nisin终浓度为25 ng/mL，继续在30℃诱导培养5h。培养结束后10000 rpm离心5 min，培养基上清用于HPLC-MASS检测。
2、HPLC-MASS检测GLP-1在乳酸乳球菌中的诱导分泌表达
将培养后的上清按要求进行纯化后，进行HPLC-MASS检测。
实施例4：GLP-1、mGLP-1蛋白体外DPP-IV酶切降解实验
为了验证mGLP-1是否可以抵抗DPP-IV酶切降解，延长半衰期，我们合成了GLP-1、mGLP-1多肽(上述氨基酸序列由南京金斯瑞生物工程有限公司合成)。用纯化的人DPP-IV蛋白酶(ProSpec，#enz-375)对上述GLP-1、mGLP-1蛋白进行了体外酶解实验。结果显示，如图8所示，mGLP-1的稳定性显著增加，相对于GLP-1，稳定性增加了50％。表明本发明提供的mGLP-1可以显著抵抗DPP-IV的降解作用，在体内有更大的潜力发挥治疗 效果。
实施例5：NZ3900-GLP-1菌株，NZ3900-mGLP-1菌株治疗Db/Db小鼠实 验
选择八周龄、雄性B6-Db/Db肥胖小鼠(所述小鼠购自南京大学模式动物研究所)作为实验对象，实验前根据体重和血糖平均分为三个组，每组8只。小鼠自由饮食一段时间(饲喂10Kcal％低脂饲料，Research diet，D12450B)。其中口服GLP-1、mGLP-1组(口服菌数剂量为1X1012NZ3900-GLP-1、NZ3900-mGLP-1菌株/天/次)，对照组(口服菌数剂量为1X1012 NZ3900空载体菌株/天/次)。其中，图9为口服GLP-1、mGLP-1对小鼠葡萄糖耐受水平的影响。结果表明，在起始体重和空腹血糖相等的情况下，连续灌胃口服GLP-1、mGLP-1三周之后，mGLP-1处理组Db/Db小鼠的葡萄糖耐受得到极显著提高，效果明显优于GLP-1处理组。上述结果表明，本发明构建的NZ3900-mGLP-1菌株可以通过口服的方式达到提高糖尿病小鼠葡萄糖敏感性，改善糖尿病进程的显著疗效。

Claims (8)

  1. 一种GLP-1类似物mGLP-1，所述mGLP-1的序列如SEQ ID NO:4所示；
  2. 编码如权利要求1所述的mGLP-1的核苷酸序列；
    优选地，所述核苷酸序列如SEQ ID NO:2所示。
  3. 一种表达GLP-1的重组表达载体，其在表达载体pNZ8149插入信号肽、增强子序列、肠激酶识别位点、人源GLP-1的核苷酸序列或人源mGLP-1的核苷酸序列或其相关衍生物；
    优选地，所述信号肽为spUSP45；更优选地，所述信号肽的序列如SEQ ID NO:5所示；
    优选地，所述增强子序列为LEISS；
    优选地，所述肠激酶识别位点为DDDDK。
  4. 根据权利要求3所述的重组表达载体，其特征在于，所述肠激酶识别位点被存在于人十二指肠粘膜中的肠激酶识别并切割进而产生与机体内源序列相同的蛋白；
    优选地，所述信号肽插入到表达载体pNZ8149的多克隆位点NcoI和SacI之间；
    优选地，所述人源GLP-1的核苷酸序列如SEQ ID NO:1所示；
    优选地，所述人源mGLP-1的核苷酸序列如SEQ ID NO:2所示；
    优选地，所述衍生物为编码所述GLP-1以及mGLP-1的相关突变体，类似物以及激动剂的核苷酸序列。
  5. 一种分泌GLP-1的蛋白表达系统，所述表达系统为被如权利要求3或4所述的重组表达载体转化的肠道正常菌；
    优选地，所述肠道正常菌选自乳酸杆菌、双歧杆菌或乳酸菌；
    更优选地，所述乳酸菌选自嗜酸乳杆菌(Lactobacillus acidophilus)、干酪乳杆菌(Lactobacillus casei)、卷曲乳杆菌(Lactobacillus crispatus)、保加利亚乳杆菌(Lactobacillusbulgaricus)、德氏乳杆菌(Lactobacillus delbrueckii)、发酵乳杆菌(Lactobacillus fermentium)、格氏乳杆菌(Lactobacillus gasseri)、瑞士乳杆菌(Lactobacillus helveticus)、约氏乳杆菌(Lactobacillus johnsonii)、副干酪乳 杆菌(Lactobacillus paracasei)、植物乳杆菌(Lactobacillusplantarum)、罗伊氏乳杆菌(Lactobacillus reuteri)、鼠李糖乳杆菌(Lactobacillus rhamnosus)、唾液乳杆菌(Lactobacillus salivarius)，以及乳酸乳球菌乳酸亚种(Lactococcuslactis subspecies lactis)等；
    更优选地，所述乳酸菌为L.1actis NZ3900；
    更进一步优选地，所述分泌GLP-1的蛋白表达系统为乳酸菌NICE(the nisin controlled expression)系统。
  6. 如权利要求5所述的分泌GLP-1的蛋白表达系统的制备方法，其包括以下步骤：
    1)人工合成GLP-1类似物mGLP-1、GLP-1或编码所述GLP-1或mGLP-1的相关突变体，类似物以及激动剂的核苷酸序列，上下游酶切位点分别为NcoI和SacI；
    优选地，人工合成序列NcoI-SPusp45-LEISS-DDDDK-GLP-1-SacI或NcoI-SPusp45-LEISS-DDDDK-mGLP-1-SacI；
    2)将步骤1)合成的序列插入表达载体pNZ8149中，构建表达GLP-1的重组表达载体；
    3)将步骤2)的重组表达载体转化至肠道正常菌中；
    4)将步骤3)得到的重组肠道正常菌接种于培养基中培养表达。
  7. 一种药物组合物，所述药物组合物包括治疗有效量的如权利要求1所述的GLP-1类似物mGLP-1、如权利要求2所述的核苷酸序列、如权利要求3或4所述的表达GLP-1的重组表达载体或如权利要求5所述的分泌GLP-1的蛋白表达系统；
    优选地，所述药物组合物为口服药物组合物；
    优选地，所述药物组合物还包括药学上可用的载体和/或辅料。
  8. 如权利要求1所述的GLP-1类似物mGLP-1、如权利要求2所述的核苷酸序列以及GLP-1核苷酸序列改变物、如权利要求3或4所述的表达GLP-1的重组表达载体或如权利要求5所述的分泌GLP-1的蛋白表达系统在制备药物中的用途；
    优选地，所述药物为预防和/治疗糖尿病、肥胖、糖尿病肾损伤、糖尿病大血管病变、糖尿病微血管损伤、糖尿病性视网膜病变、胰高血糖素血症、坏死性游走性红斑、高血糖素瘤等代谢性疾病、冠心病、高血压性心脏病、 瓣膜性心脏病、酒精性心肌病、糖尿病心血管并发症以及用于治疗阿尔兹海默和帕金森等神经损伤性疾病的药物。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114426942A (zh) * 2022-01-25 2022-05-03 中国科学院动物研究所 一种重组乳酸乳球菌、微胶囊及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101535341A (zh) * 2006-07-18 2009-09-16 森托科尔奥索生物科技公司 人glp-1模拟体、组合物、方法和用途
WO2011071957A1 (en) * 2009-12-07 2011-06-16 Sea Lane Biotechnologies, Llc Conjugates comprising an antibody surrogate scaffold with improved pharmacokinetic properties
CN102329766A (zh) * 2011-09-16 2012-01-25 暨南大学 可用于预防或治疗人溃疡性结肠炎的重组食品级乳酸菌、其制备方法与应用
CN102796755A (zh) * 2012-06-28 2012-11-28 郑州大学 一种乳酸乳球菌表达载体及其制备方法与应用
CN103804488A (zh) * 2012-11-15 2014-05-21 刘占良 使用食品级乳酸菌生产和递送的胰高血糖素样肽(glp-1和glp-2)及其制备方法
CN106011165A (zh) * 2016-05-06 2016-10-12 南昌大学 分泌表达glp-1的乳酸乳球菌的制备方法及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101535341A (zh) * 2006-07-18 2009-09-16 森托科尔奥索生物科技公司 人glp-1模拟体、组合物、方法和用途
WO2011071957A1 (en) * 2009-12-07 2011-06-16 Sea Lane Biotechnologies, Llc Conjugates comprising an antibody surrogate scaffold with improved pharmacokinetic properties
CN102329766A (zh) * 2011-09-16 2012-01-25 暨南大学 可用于预防或治疗人溃疡性结肠炎的重组食品级乳酸菌、其制备方法与应用
CN102796755A (zh) * 2012-06-28 2012-11-28 郑州大学 一种乳酸乳球菌表达载体及其制备方法与应用
CN103804488A (zh) * 2012-11-15 2014-05-21 刘占良 使用食品级乳酸菌生产和递送的胰高血糖素样肽(glp-1和glp-2)及其制备方法
CN106011165A (zh) * 2016-05-06 2016-10-12 南昌大学 分泌表达glp-1的乳酸乳球菌的制备方法及其应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114426942A (zh) * 2022-01-25 2022-05-03 中国科学院动物研究所 一种重组乳酸乳球菌、微胶囊及其应用

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