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WO2020118843A1 - 一种glp-1突变体及其制备方法和用途 - Google Patents

一种glp-1突变体及其制备方法和用途 Download PDF

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WO2020118843A1
WO2020118843A1 PCT/CN2019/071736 CN2019071736W WO2020118843A1 WO 2020118843 A1 WO2020118843 A1 WO 2020118843A1 CN 2019071736 W CN2019071736 W CN 2019071736W WO 2020118843 A1 WO2020118843 A1 WO 2020118843A1
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solution
recombinant
seq
bacteria
nucleotide sequence
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PCT/CN2019/071736
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English (en)
French (fr)
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顾建文
马婕
马永平
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四川利通科创生物医药科技有限公司
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Priority claimed from CN201811521016.1A external-priority patent/CN110251662B/zh
Priority claimed from CN201811520996.3A external-priority patent/CN110251661B/zh
Priority claimed from CN201811519742.XA external-priority patent/CN110256553B/zh
Application filed by 四川利通科创生物医药科技有限公司 filed Critical 四川利通科创生物医药科技有限公司
Publication of WO2020118843A1 publication Critical patent/WO2020118843A1/zh

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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
    • 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

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to a GLP-1 mutant and its preparation method and use.
  • T2DM Type 2 Diabetes Mellitus
  • overweight and obesity will further aggravate the insulin resistance of T2DM patients, and greatly increase the risk of cardiovascular disease and other complications.
  • Strengthening weight loss can bring clear benefits to patients with T2DM: reducing insulin resistance, repairing damaged islet ⁇ -cell function, optimizing blood sugar control, and improving risk factors related to cardiovascular disease.
  • the vicious circle caused by obesity and T2DM makes the traditional treatment mode with "hypoglycemic" as the core into a dilemma, which also makes more and more scholars pay attention to the importance of "weight loss” treatment.
  • weight loss drugs bring good weight loss efficacy, they gradually withdraw from the market due to serious safety problems.
  • the weight loss drugs currently in clinical use are mainly orlistat capsules approved in 1999, lorcaserin and phentermine and topiramate capsules approved in 2012, and naltrexone hydrochloride and bupropion compound sustained release approved in 2014. Tablets and liraglutide injection.
  • Liraglutide is a glucagon-like peptide-1 (Glucagon-likepeptide-1, GLP-1)-like peptide and is a new type of drug based on the action of incretin.
  • GLP-1 analogue peptides mainly reduce the energy intake of patients by inhibiting central appetite for appetite and inhibiting gastrointestinal motility and increasing satiety, thereby exerting the effect of weight loss.
  • Linaclutide has a glucose concentration-dependent hypoglycemic effect, and monotherapy does not cause hypoglycemia.
  • Liraglutide was launched in the United States and the European Union in 2009 and 2010 for the treatment of type 2 diabetes (T2DM).
  • T2DM type 2 diabetes
  • the FDA and the European Medicines Agency approved it as a supplement to diet control and physical exercise for the treatment of chronic obesity.
  • the effect is better than the mainstream weight loss drug on the market-orlistat.
  • the weight loss effect is clear.
  • Liraglutide was approved for listing in China in 2011. Long-term trials have shown that liraglutide can effectively reduce the weight of patients.
  • the suitable population is adults with a body mass index (BMI) greater than or equal to 30, or a BMI of 27 or more, accompanied by T2DM, hypertension, or high cholesterol. An adult with complications of obesity.
  • BMI body mass index
  • GLP-1 like peptide polypeptide drugs including liraglutide need to be administered by injection.
  • Direct oral administration is ineffective, which has the disadvantages of inconvenience.
  • the treatment of diabetes usually requires long-term, continuous medication.
  • the method of injection administration is inconvenient to use and carry, and the medication compliance is poor.
  • oral preparations are most in line with people's habit of "taking" medicines, and the preparation process is mature and simple. Therefore, it is of great significance to start by changing the route of administration of GLP-1 analogue peptides, to develop medicines and to carry them conveniently, and to be suitable for long-term use of GLP-1 analogue peptides.
  • the purpose of the present invention is to provide a GLP-1 mutant and its preparation method and use.
  • the present invention provides a GLP-1 mutant, which is obtained on the basis of wild-type GLP-1 through amino acid site mutation, and its amino acid sequence is shown in SEQ ID NO: 1.
  • the invention also provides a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1.
  • the present invention provides a recombinant vector comprising the above nucleotide sequence; further, the recombinant vector is a recombinant prokaryotic vector; further, the prokaryotic vector is a pGEX plasmid or a pMG36e vector.
  • the present invention provides a recombinant bacterium, characterized in that it contains the above-mentioned recombinant vector; further, the recombinant bacterium is recombinant E. coli Nissle 1917 or recombinant lactic acid bacterium.
  • the present invention provides a method for preparing the above-mentioned GLP-1 mutant, which includes the following steps:
  • the medium is LB medium
  • the medium is MRS medium.
  • the present invention provides the use of the above mutants, nucleotide sequences, recombinant vectors, and recombinant bacteria in the preparation of a medicament for treating diabetes.
  • the present invention provides a medicament for treating diabetes, which contains the above mutant, nucleotide sequence, recombinant vector and/or recombinant bacteria.
  • the present invention provides the use of the above mutants, nucleotide sequences, recombinant vectors, and recombinant bacteria in the preparation of drugs for treating obesity.
  • the invention also provides a medicine for weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2 Recombinant vectors of nucleotide sequences and/or recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 are effective ingredients.
  • the invention also provides a health food for weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2
  • SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO: 2
  • the recombinant vector of nucleotide sequence and/or the recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2 is the active ingredient.
  • the invention provides a medicament for treating diabetes or weight loss, which has an amino acid sequence as shown in SEQ ID NO: 1 GLP-1 mutant, SEQ ID NO: 2 nucleotide sequence, including SEQ ID NO:
  • the preparation is an oral preparation.
  • the oral preparation is a tablet, capsule, powder, granule or oral liquid.
  • the powder is a lyophilized powder.
  • the lyophilized powder is prepared according to the following method: take the bacterial precipitate of the recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2, and resuspend it with a 20% (w/v) oligofructose solution Bacteria, then add 15% (w/v) glycerol solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and 3% (w /v) trehalose solution, prepared into a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, lyophilized after filling, and the mixed liquid contains fructooligosaccharide solution, glycerin solution, and skimmed milk powder
  • the volume ratio of the solution, lactose solution and trehalose solution is (1-10): (1-10): (1-10): (1-10), preferably 2:3:5: 6:1; preferably, the number of viable bacteria per gram of mixed liquid
  • the powder is a dry powder
  • the dry powder according to the following method: take the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2, add a low concentration of 20% (w/v) Resuspend the bacteria in the polyfructose solution, then add 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and 3% (w/v) trehalose solution.
  • the preparation is a granule
  • the granule is prepared according to the following method: the bacterial precipitate of the recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times The weight of magnesium stearate is mixed and granulated to obtain granules; in the mixed liquid, the volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and
  • the preparation is a tablet, and the tablet is as follows: take the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2, and add a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times The weight of magnesium stearate is mixed and compressed to obtain tablets.
  • the volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and trehalose solution is (1-10): (1-10): (1-10): ( 1-10): (1-10), preferably 2:3:5:6:1; preferably, the number of viable bacteria per gram of mixed liquid is 10 10 cfu.
  • the preparation is a capsule
  • the capsule is prepared according to the following method: a bacterial precipitate of a recombinant bacterium containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution, then add 15% (w/v) glycerin solution, 10% (w/v) skim milk powder solution, 3% (w/v) lactose solution and concentration It is a 3% (w/v) trehalose solution, prepared as a mixed liquid of 10 10 ⁇ 10 12 cfu live bacteria per gram, dried to obtain a powder, and added 0.5 to 2 times the weight of flavoring agent, 0.5 to 2 times Weight magnesium stearate, mixed, granulated to obtain granules, filled into capsules, obtained capsules; volume ratio of fructooligosaccharide solution, glycerin solution, skimmed milk powder solution, lactose solution and tre
  • the preparation is an oral liquid
  • the oral liquid is taken as follows: the bacterial precipitate of recombinant bacteria containing the nucleotide sequence shown in SEQ ID NO: 2 is added at a low concentration of 20% (w/v) Resuspend bacteria in polyfructose solution or L-arabinose solution with a concentration of 2% (w/v), prepare a fresh bacterial solution of not less than 10 10 ⁇ 10 12 cfu of live bacteria per milliliter, fill it, preferably; The number of viable bacteria per gram of mixed liquid is 10 10 cfu.
  • the GLP-1 mutant of the present invention reduces the degradation of DPP-IV, and has a half-life of about 3 hours; and the recombinant probiotic bacteria containing the GLP-1 mutant constructed by the present invention can be prepared It can be administered into various types of solid and liquid preparations to achieve oral administration to avoid the pain of long-term injection of patients.
  • the genetically engineered probiotic bacteria can survive and colonize the human intestine and become a functional in vivo bioreactor.
  • the continuous production and secretion of GLP-1 mutant polypeptides can play a role in continuous hypoglycemic, and the clinical application prospects are good.
  • FIG. 1 Screening results of GLP-1GM mutant protein in the present invention.
  • FIG. 1 SDS-PAGE and WB detection results of the GLP-1GM mutant of the present invention.
  • FIG. 3 Glucose-lowering effect of GLP-1GM mutant expressed in E. coli in the present invention by intragastric administration of T2DM rat model.
  • FIG. 4 Glucose-lowering effect of GLP-1GM mutant expressed by lactic acid bacteria in the present invention by intragastric administration of T2DM rat model.
  • Fig. 5 The effect of mutant GM transformation of E. coli Nissle1917 on the body weight and weight gain value of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 6 The effect of mutant GM transformation of Escherichia coli Nissle1917 on the food intake of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 7 The effect of mutant GM transformed Escherichia coli Nissle1917 on the area under the fasting blood glucose and blood glucose concentration change curve of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 8 The effect of GM transformation of mutant E. coli Nissle1917 on the weight of adipose tissue around the kidney and the ratio of liposomes in mice, compared with the normal group, ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 9 The effect of GM transformation of mutant E. coli Nissle1917 on the weight of adipose tissue and the ratio of liposomes around the testes (ovaries) of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 10 The effect of mutant GM transformation of E. coli Nissle1917 on the liver weight of mice, compared with the normal group ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01; compared with the model group *p ⁇ 0.05, **p ⁇ 0.01.
  • pGEX-4T-1, pMG36e, pET32a, E.coli TOP10 and E.coli BL21 (DE3), E. coli Nissle 1917, Lactobacillus, Lactococcus and other strains and plasmids are from Chengdu Lilai Biotechnology Co., Ltd. and Chongqing Medical University Preserved by the Department of Biochemistry and Molecular Biology; male rats are provided by the Laboratory Animal Center of Chongqing Medical University.
  • T4 ligase Taq common enzyme, SalI, BamHI, protein Marker, DNA Marker, Plasmid Mini Kit I, Cycle-Pure Kit, gel recovery kit, IPTG, erythromycin, ampicillin, urethromycin, BCA protein concentration Reagents such as measurement kits and PMSF (all commercially available).
  • GM is based on the original sequence of GLP-1, mutating the 8th alanine of GLP-1 to histidine, the 27th glutamic acid to lysine, and the 34th lysine to arginine. Acid, arginine at position 36 was mutated to glycine, glycine at position 37 was mutated to lysine, and a sequence was added at the end.
  • C33 is based on the original sequence of GLP-1, mutating glutamic acid at position 9 of GLP-1 to cysteine and valine at position 33 to cysteine.
  • K34 is based on the original sequence of GLP-1, mutating GLP-1 glycine at position 35 to lysine, arginine at position 36 to lysine, and glycine at position 37 to lysine.
  • R34 is based on the original GLP-1 sequence, mutating the 34th lysine to arginine, the 35th glycine to arginine, and the 37th glycine to arginine.
  • Natural GLP-1 is easily hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV), and its half-life is less than 5 minutes, which is not suitable for clinical application, so this animal experiment was not conducted.
  • DPP-IV dipeptidyl peptidase IV
  • GLP-1GM GLP-1 mutant numbered GM
  • Example 2 Expression and activity verification of recombinant E. coli of the mutant of the present invention
  • the pGEX-GLP-1GM expression vector was transformed into E. coli Nissle 1917, and PCR and sequencing confirmed that pGEX-GLP-1GM was successfully transformed.
  • Recombinant Escherichia coli group Nissle 1917 was inoculated onto LB medium supplemented with ampicillin at a final concentration of 100 ⁇ g/ml, and cultured on a shaker at 37°C and 250 r/min until the OD 600 value reached 0.8 to 1.0. Discard the supernatant and adjust the OD 600 value to 1.2 with PBS.
  • the GLP-1GM mutant expressed in E. coli Nissle 1917 has a hypoglycemic effect 2 hours after intragastric administration (compared to the control group without intragastric administration, P ⁇ 0.01). After 8 hours of intragastric administration, the blood glucose slightly increased.
  • Escherichia coli Nissle1917 (prepared in Example 2) stably transformed with genes identified by PCR verification and induced expression experiments was inoculated at 37°C, 250r /min shaker culture until the OD600 value reaches 0.8 ⁇ 1.0, centrifuge for 5min to harvest bacteria, wash twice with physiological saline, centrifuge to collect bacterial pellets, add 2% (w/v) concentration of L-arabinose solution or The 20% oligofructose solution was used to resuspend the bacteria, and it was prepared into a fresh bacterial solution of 10 10 cfu live bacteria per ml. After filling, it was stored in a refrigerator at 4°C.
  • the flavoring agent includes sweeteners or fruit flavoring aromatics, mix evenly for 20-30 minutes, put into dry granulation mechanism and collect After the 10-100 mesh particles were packed, the transformed E. coli Nissle 1917 granules were obtained.
  • the prepared E. coli Nissle1917 granules are filled into hollow hard capsules according to the corresponding specifications, and the resulting capsules are placed in a polishing machine to throw away excess dust to obtain transformed E. coli
  • Mutant GM transforms lactic acid bacteria
  • MRS liquid culture medium supplemented with erythromycin at a concentration of 20 ⁇ g/ml was steam sterilized, and lactic acid bacteria (prepared in Example 5) stably transformed with genes identified by PCR verification and induced expression experiments were inoculated, and incubated at 30°C until the OD600 value When it reaches 0.8 ⁇ 1.0, centrifuge, discard the supernatant, take the bacterial pellet and add 2% (w/v) L-arabinose solution or 20% fructooligosaccharide solution to resuspend the bacteria, and make it not lower per ml Fresh bacterial solution at 10 10 viable bacteria was refrigerated and stored in a refrigerator at 4°C.
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria has a certain hypoglycemic effect. Similar to the results in E. coli, the half-life of GLP-1GM is still short, and the blood glucose concentration decreases after 2 hours of intragastric administration (P ⁇ 0.01), After 5-7 hours, the blood glucose increased slightly (P ⁇ 0.05), and the hypoglycemic effect was weakened at 24h. If the same amount of recombinant lactic acid bacteria was immediately gavaged twice, the hypoglycemic effect was obtained again after 2h (ie 26h) (P ⁇ 0.01).
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria also has an oral hypoglycemic effect.
  • the GLP-1GM mutant expressed in recombinant lactic acid bacteria also has an oral hypoglycemic effect.
  • the GLP-1 mutant of the present invention reduces the degradation of DPP-IV, and the half-life is about 3h; and the recombinant probiotic bacteria containing the GLP-1 mutant constructed by the present invention has a continuous decrease Sugar effect.
  • SPF grade Kunming mice weighing 20-25g, were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. Certificate number: SCXK (Chuan) 2015-030. Animals were inspected for quarantine and adaptability for 3 days after acceptance, and the experiment was started after passing. Animals can drink water and eat freely, temperature (20 ⁇ 2)°C, humidity (60 ⁇ 5)%, 12h light period.
  • the experimental animals were randomly divided into: normal group, model group, recombinant E. coli Nissle 1917 (prepared in Example 5) high, middle and low dose groups, 10 animals in each group.
  • the normal group was fed with ordinary feed, and the remaining four groups were fed with high-fat and high-sugar feed (feed formula: 67% for ordinary feed, 20% for sucrose, 10% for lard, 2% for cholesterol, and 1% for sodium cholate).
  • the test animals eat and drink freely every day.
  • the recombinant Escherichia coli Nissle1917 high-medium-low-dose group was gavaged with 10 10 , 10 9 , and 10 8 CFU bacterial fluids, respectively, and the model group was gavaged with an equal volume of sterile saline, once a day, for the fourth week. And the eighth week of material determination.
  • Body weight Determine the body weight of experimental animals at a fixed time every week, and calculate the weight gain value.
  • the body weight and weight gain values of the model group were significantly higher than those of the normal group (p ⁇ 0.05).
  • the high-medium and low-dose groups of bacterial solution can significantly reduce the body weight and weight gain of experimental animals at four weeks (p ⁇ 0.05), and the high-medium-dose bacterial group at eight weeks can significantly reduce the weight and weight gain of experimental animals. Value (p ⁇ 0.05).
  • the high-medium-low-dose group of bacteria solution can significantly reduce the food intake of experimental animals (p ⁇ 0.05). Compared with the normal group, there was no significant difference in food intake in the high-medium-low-dose group (p>0.05).
  • the fatty tissue and liposomes around the kidneys in the model group were significantly higher than those in the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group can significantly reduce the fatty tissue and liposome ratio around the kidneys of experimental animals (p ⁇ 0.05); at the eighth week, the high-dose bacterial liquid group can be significantly reduced Adipose tissue and liposome ratio around kidneys of experimental animals (p ⁇ 0.05).
  • the adipose tissue and liposomes around the testes (ovaries) in the model group were significantly higher than those in the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group at the fourth week can significantly reduce the ratio of adipose tissue and liposomes around the testicles (ovaries) of experimental animals (p ⁇ 0.05); at the eighth week, the high-dose bacterial liquid group can be significantly Reduce the ratio of adipose tissue and liposomes around the testis (ovaries) of experimental animals (p ⁇ 0.05).
  • the liver weight of the model group was significantly higher than that of the normal group (p ⁇ 0.05).
  • the high-dose bacterial liquid group can significantly reduce the liver weight of experimental animals (p ⁇ 0.05).
  • the mutant GM transformed into E. coli Nissle1917 can inhibit the appetite of experimental mice and reduce energy absorption; significantly reduce the body weight, body fat content, liposome ratio and liver weight of experimental mice, with very obvious weight loss and lipid-lowering The role.
  • the transformed Escherichia coli Nissle1917 can also significantly reduce the area under the curve of blood glucose changes in the oral glucose tolerance test, and has a certain tendency to lower blood glucose.

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Abstract

本发明公开了一种GLP-1突变体。本发明还公开了编码GLP-1突变体的核苷酸序列，及包含该核苷酸序列的重组载体、重组菌，还公开了所述GLP-1突变体的制备方法和用途，本发明还提供了用于治疗糖尿病或者减肥的药物。本发明包含GLP-1突变体的药物，可以有效降低血糖浓度或减肥降脂，可用于治疗II型糖尿病或肥胖症，临床应用前景良好。

Description

一种GLP-1突变体及其制备方法和用途 技术领域
本发明属于基因工程技术领域，具体涉及一种GLP-1突变体及其制备方法和用途。
背景技术
随着人们膳食结构和生活方式的改变，肥胖已成为世界范围内的流行病。目前全球范围内超重和肥胖的发生率都呈逐年增加的趋势。特别是我国，近年来超重和肥胖的发生率已接近甚至达到发达国家初期的水平。对于那些BMI指数高的人群来说，肥胖会引起其他一些疾病发病率的上升，其中最为显著的是糖尿病、心血管疾病和癌症，超重/肥胖严重危害人类的健康。
同时，超重/肥胖是2型糖尿病(Type 2Diabetes Mellitus，T2DM)发生、发展的独立危险因素，两者是相伴相生的疾病状态。研究表明，体质指数每增加1kg/m 2，T2DM的患病风险就增加12％；而在T2DM患者中，超重和肥胖症的患病率超过80％。同时，超重和肥胖状态会进一步加重T2DM患者的胰岛素抵抗状态，并大大增加心血管疾病等并发症的发生风险。强化减肥可为T2DM患者带来明确的收益：减轻胰岛素抵抗状态、修复受损的胰岛β细胞功能、优化血糖控制及改善心血管疾病的相关危险因素等。肥胖与T2DM引发的恶性循环状态，使传统的以“降糖”为核心的治疗模式进入到一个两难的境地，这也使越来越多学者开始关注“减肥”治疗的重要性。
单纯生活方式干预虽然能使肥胖患者的体重下降，但长期疗效并不理想，究其原因，一方面与大部分患者很难严格坚持减肥生活方式有关；另一方面还与大部分传统药物在应用过程中均会不可避免地增加体重有关。
但是，多种减肥药物在带来良好减肥疗效的同时，因存在着严重的安全问题逐步退出市场。目前临床应用的减肥药品主要有1999年批准的奥利司他胶囊，2012年批准的氯卡色林和苯丁胺和托吡酯胶囊，2014年批准的盐酸纳曲酮、安非他酮复方缓释片以及利拉鲁肽注射液。
利拉鲁肽(Liraglutide)是胰高血糖素样肽1(glucagon-likepeptide-1，GLP-1)类似肽，为新型的基于肠促胰素作用的药物。GLP-1类似肽主要通过抑制中枢摄食欲望和抑制胃肠蠕动，增加饱腹感来减少患者对能量的摄入，从而发挥减肥的疗效。利那鲁肽具有葡萄糖浓度依赖性的降糖作用，单药治疗不会导致低血糖。
利拉鲁肽于2009年和2010年分别在美国和欧盟上市用于2型糖尿病(T2DM)的治疗。2014年和2015年，FDA和欧洲药品管理局批准其作为饮食 控制和体育锻炼的补充，用于慢性肥胖的治疗，效果优于市场上的主流减肥药—奥利司他，减肥效果明确，是用于肥胖治疗的首款日用一次的GLP-1类似肽。利拉鲁肽于2011年在中国批准上市。长期试验表明，利拉鲁肽能够有效减轻患者体重，其适应人群是体重指数(BMI)大于或等于30的成年人，或者是BMI为27及以上、伴有T2DM、高血压或高胆固醇等至少一种肥胖并发症的成年人。在人们强烈的减肥欲望与疗效不佳的现有减肥药形成大矛盾的今天，开发GLP-1类似肽类的减肥药也是一个不二的选择。因此，兼具减肥和降血糖双重效益的药物具有十分广阔的市场前景。
目前包括利拉鲁肽在内的GLP-1类似肽多肽药物，均需注射给药，直接口服无效，存在用药不便等缺点。但是糖尿病的治疗通常需要长期、持续用药。注射给药的方式，使用携带不方便，用药依从性差，还可能存在刺激性和过敏反应等风险，给患者带来巨大的生理和心理痛苦。而口服制剂最符合人们“服”药的习惯，制剂工艺也成熟而简单。因此从改变GLP-1类似肽的给药途径入手，研制用药和携带方便，适用于长期使用的GLP-1类似肽类药物，意义重大。
发明内容
本发明的目的在于提供一种GLP-1突变体及其制备方法和用途。
本发明提供了一种GLP-1突变体，它是在野生型GLP-1基础上，经过氨基酸位点突变得到，它的氨基酸序列如SEQ ID NO：1所示。
本发明还提供了编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
其中，序列如SEQ ID NO：2所示。
本发明提供了一种重组载体，它包含上述核苷酸序列；进一步地，所述重组载体是重组原核载体；更进一步地，所述原核载体为pGEX质粒或者pMG36e载体。
本发明提供了一种重组菌，其特征在于：它包含上述重组载体；进一步地，所述重组菌为重组大肠杆菌Nissle 1917或者重组乳酸菌。
本发明提供了一种制备上述GLP-1突变体的方法，包含如下步骤：
取上述重组菌，接种到LB培养基上，振荡培养至OD 600为0.8-1.0时，离心，取沉淀，分离纯化，即可。
其中，当重组菌为大肠杆菌Nissle 1917时，培养基为LB培养基；
重组菌为乳酸菌时，培养基为MRS培养基。
本发明提供了上述突变体、核苷酸序列、重组载体、重组菌在制备治疗糖尿病的药物中的用途。
本发明提供了一种治疗糖尿病的药物，它含有上述突变体、核苷酸序列、重组载体和/或重组菌。
本发明提供了上述突变体、核苷酸序列、重组载体、重组菌在制备治疗肥胖的药物中的用途。
本发明还提供了一种减肥的药物，它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分。
本发明还提供了一种减肥的保健食品，它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分。
本发明提供了一种用于治疗糖尿病或者减肥的药物，它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂；所述制剂为口服制剂。
优选地，所述口服制剂为片剂、胶囊剂、粉剂、颗粒剂或口服液。
优选地，所述粉剂为冻干粉剂。
进一步地，所述冻干粉按照如下方法制备：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，分装后冻干，即可；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
优选地，所述粉剂为干燥粉剂，所述干燥粉剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 11cfu活菌的混合液体，烘干，烘干的温度不超过40℃；所述混合液体中，低聚果糖溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)，优选为2：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
优选地，所述制剂为颗粒剂，所述颗粒剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果 糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，制粒，得颗粒剂；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
优选地，所述制剂为片剂，所述片剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，压片，得片剂。
优选地，所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
优选地，所述制剂为胶囊剂，所述胶囊剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，制粒，得颗粒剂，装胶囊，得胶囊剂；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
优选地，所述制剂为口服液，所述口服液按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液或者浓度为2％(w/v)的L-阿拉伯糖溶液重悬细菌，配制成每毫升不低于10 10～10 12cfu活菌的新鲜菌液，灌装，即可；优选地，所述每克混合液体中的活菌数量为10 10cfu。
与天然的GLP-1相比，本发明GLP-1突变体，减小了DPP-Ⅳ的降解作用，半衰期约为3h；而且本发明构建的含有GLP-1突变体的重组益生菌，可制备成多种类型的固体及液体制剂实现口服给药，避免患者长期注射用药的 痛苦；同时，人体服用后，该基因工程益生菌可以在人体肠道中存活定植，成为功能性的体内生物反应器，持续产生并分泌GLP-1突变体多肽，从而起到持续降糖作用，临床应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1本发明中GLP-1GM突变体蛋白的筛选结果。
图2本发明的GLP-1GM突变体SDS-PAGE及WB检测结果。
图3本发明中大肠杆菌表达的GLP-1GM突变体灌胃T2DM大鼠模型的降糖效果。
图4本发明中乳酸菌菌表达的GLP-1GM突变体灌胃T2DM大鼠模型的降糖效果。
图5突变体GM转化大肠杆菌Nissle1917对小鼠体重和体重增长值的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
图6突变体GM转化大肠杆菌Nissle1917对小鼠摄食量的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
图7突变体GM转化大肠杆菌Nissle1917对小鼠空腹血糖及血糖浓度变化曲线下面积的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
图8突变体GM转化大肠杆菌Nissle1917对小鼠肾脏周围脂肪组织重量及脂体比的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
图9突变体GM转化大肠杆菌Nissle1917对小鼠睾丸(卵巢)周围脂肪组织重量及脂体比的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
图10突变体GM转化大肠杆菌Nissle1917对小鼠肝脏重量的影响，与正常组相比△p＜0.05，△△p＜0.01；与模型组相比*p＜0.05，**p＜0.01。
具体实施方式
下面以实施例作进一步说明，但本发明不局限于这些实施例。
本发明所用的实验试剂与仪器如下：
pGEX-4T-1、pMG36e、pET32a、E.coli TOP10及E.coli BL21(DE3)、大肠杆菌Nissle 1917、乳酸杆菌、乳酸乳球菌等菌株与质粒由成都里来生物科技有限公司和重庆医科大学生物化学与分子生物学教研室保存；雄性大鼠由重庆医科大学实验动物中心提供。T4连接酶、Taq普通酶、SalⅠ、BamHⅠ、蛋白质Marker、DNA Marker、Plasmid Mini kit I、Cycle-Pure Kit、胶回收试剂盒、IPTG、红霉素、氨苄青霉素、脲佐菌素、BCA蛋白浓度测定试剂盒、PMSF等试剂(均为市售)。
实施例1本发明突变体序列的筛选及活性检测
从GLP-1原始序列出发，改造设计了4个突变体多肽序列，通过化学合成(中肽生化)，获得纯度高达85％的多肽产物。多肽序列如下：
GLP-1片段(7-37aa)：HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
GM(SEQ ID NO：1)：
HHEGTFTSDVSSYLEGQAAKKFIAWLVRGGKKKKKYGRKKRRQRRREF
C33：HACGTFTSDVSSYLEGQAAKEFIAWLCKGRG
K34：HAEGTFTSDVSSYLEGQAAKEFIAWLVKKKK
R34：HAEGTFTSDVSSYLEGQAAKEFIAWLVRRRR
GM是在GLP-1原始序列的基础上，将GLP-1第8位丙氨酸突变为组氨酸，27位的谷氨酸突变为赖氨酸，34位的赖氨酸突变为精氨酸，36位的精氨酸突变为甘氨酸，37位的甘氨酸突变为赖氨酸，并且末端加入一段序列。
C33是在GLP-1原始序列的基础上，将GLP-1第9位谷氨酸突变为半胱氨酸，33位缬氨酸突变为半胱氨酸。
K34是在GLP-1原始序列的基础上，将GLP-1第35位甘氨酸突变为赖氨酸、第36位精氨酸突变为赖氨酸、第37位甘氨酸突变为赖氨酸。
R34是在GLP-1原始序列的基础上，将GLP-1第34位赖氨酸突变为精氨酸，第35位甘氨酸突变为精氨酸，第37位甘氨酸突变为精氨酸。
GLP-1原始序列与突变体R34、K34和C33的区别如表1所示。
表1 GLP-1及突变体的序列比较
Figure PCTCN2019071736-appb-000001
Figure PCTCN2019071736-appb-000002
Figure PCTCN2019071736-appb-000003
取雄性大鼠，每组6只，禁食12h，测定血糖后，腹腔注射20mmol/kg葡萄糖，后腹腔注射给予上述四个突变体多肽50μg/kg。给药后各时间点用三诺血糖仪(GA-3型)测定血糖。
结果如图1所示。
天然GLP-1易被二肽基肽酶Ⅳ(DPP-Ⅳ)迅速水解失活，其半衰期小于5min，不适于临床应用，故未进行该项动物实验。
可见，编号为GM的GLP-1突变体(GLP-1GM)降糖活性较好，且半衰期长达3小时，而其它GLP-1突变体降糖能力及半衰期均较差。
实施例2本发明突变体的重组大肠杆菌表达及活性验证
1、本发明GLP-1突变体的大肠杆菌表达
(1)在GLP-1突变体GM的序列前加入一段信号肽序列、在3’-端加入HIV穿膜肽序列和6个His-tag序列，构建出能够分泌表达和具有穿过细胞膜能力的GLP-1突变体，其核苷酸序列如SEQ ID NO：2所示。
SEQ ID NO：2：
Figure PCTCN2019071736-appb-000004
(2)将SEQ ID NO：3所示的核苷酸序列克隆到大肠杆菌表达载体pGEX的BamHI和SalI位点，获得pGEX-GLP-1GM载体，测序正确后转化大肠杆菌E.coli BL21(DE3)中，目的片段大小约为12KD。经含镍磁珠纯化后Western blot鉴定表达结果正确，结果如图2所示。可见，本发明成功表达了GLP-1突变体蛋白。
(3)将pGEX-GLP-1GM表达载体转化到大肠杆菌Nissle 1917中，经PCR和测序鉴定pGEX-GLP-1GM转化成功。取重组大肠杆菌组菌Nissle 1917，接种到添加有终浓度为100μg/ml的氨苄青霉素的LB培养基上，37℃、250r/min摇床培养至OD 600值达到0.8～1.0，菌液离心后弃上清，沉淀用PBS调OD 600值到1.2，即可。
3、GLP-1突变体的活性验证
按照每只大鼠口服灌胃1ml重组大肠杆菌(OD 600＝1.2)处理STZ诱导成功的T2DM大鼠模型，每组6只大鼠，在灌胃后各时间点，断尾取血，采用三诺血糖仪(GA-3型)测定一次血糖，用以确定口服GLP-1GM突变体的降糖效果。结果如图3所示。
可见，大肠杆菌Nissle 1917中表达的GLP-1GM突变体灌胃后2小时具有降糖效果(与未灌胃的对照组相比，P＜0.01)。灌胃8小时后血糖略有回升。
实施例3突变体GM转化大肠杆菌Nissle1917制剂的制备
1、突变体GM转化大肠杆菌Nissle1917口服液的制备
添加有浓度为200μg/ml的红霉素抗性LB液体培养基蒸汽灭菌后，取经PCR验证及诱导表达实验鉴定的基因稳定转化的大肠杆菌Nissle1917(实施例2制备)接种，37℃、250r/min摇床培养至OD600值达到0.8～1.0后，离心5min收获细菌，用生理盐水洗涤2次，离心收集菌体沉淀后，加浓度为2％(w/v)的L-阿拉伯糖溶液或20％的低聚果糖溶液重悬细菌，配制成每毫升为10 10cfu活菌的新鲜菌液，灌装后于4℃冰箱冷藏保存。
2、突变体GM转化大肠杆菌Nissle1917/冻干粉剂的制备
取上述方法收获的细菌沉淀，加浓度为20％(w/v)的低聚果糖重悬细菌，再加入浓度为15％(w/v)的甘油、浓度为10％(w/v)的脱脂奶粉、浓度为3％(w/v)的乳糖和浓度为3％(w/v)的海藻糖，配制成每克为10 10cfu活菌的混合液体，混合液体中，20％(w/v)的低聚果糖溶液、15％(w/v)的甘油溶液、10％(w/v)的脱脂奶粉溶液、3％的乳糖溶液、3％的海藻糖溶液的体积比为2：3：5：6：1，分装后置于冷冻干燥机上冻干，封口后4-8℃冰箱保存。
3、突变体GM转化大肠杆菌Nissle1917干燥粉剂的制备
取上述方法收获的细菌沉淀，加浓度为20％(w/v)的低聚果糖重悬细菌，再加入浓度为10％(w/v)的脱脂奶粉、3％(w/v)的乳糖和3％(w/v)的海藻糖，配制成每克10 10cfu活菌的混合液体，混合液体中，20％(w/v)的低聚果糖溶液、10％(w/v)的脱脂奶粉溶液、3％的乳糖溶液、3％的海藻糖溶液的体积比为2：5：6：1，分装后置于真空干燥仪上烘干，最高温度不超过40℃， 封口后4-8℃冰箱保存。
4、突变体GM转化大肠杆菌Nissle1917颗粒剂的制备
将制得的转化大肠杆菌Nissle1917冻干粉/干燥粉剂加入0.5～2份矫味剂，矫味剂包括甜味剂或水果味芳香剂，均匀混合20-30min，投入干法制粒机制粒，收集10-100目的颗粒分装后得到转化大肠杆菌Nissle1917颗粒剂。
5、突变体GM转化大肠杆菌Nissle1917片剂的制备
将上述制得的大肠杆菌Nissle1917冻干粉与0.5～2份矫味剂、0.5～2份硬脂酸镁置于粉体混合机中混合5～15min，混合均匀后得到可直接压片的原料，置于单冲式压片机压片，得到转化的大肠杆菌Nissle1917片剂。
6、突变体GM转化大肠杆菌Nissle1917胶囊剂的制备
将制得的大肠杆菌Nissle1917颗粒，将该颗粒按相应规格灌装于空心硬胶囊中，将所得胶囊置于抛光机内抛去多余粉尘即得到转化的大肠杆菌
Nissle1917胶囊剂。
实施例4突变体GM转化乳酸菌
1、突变体GM转化乳酸菌
(1)将GLP-1GM，即SEQ ID NO：2所示的核苷酸序列构建到乳酸菌表达载体pMG36e质粒上SalI和HindIII位点，电转化乳酸杆菌后，挑取重组乳酸杆菌的单菌落，培养后提取质粒，PCR检验和测序检测GLP-1GM基因存在于该受体菌中。
(2)将上述的重组乳酸菌接种到添加有浓度为20μg/ml的红霉素的MRS培养基上，30℃静置培养至OD 600值达到0.8～1.0时，离心，弃上清，取菌体，备用。
实施例5突变体GM转化乳酸菌制剂的制备
1、突变体GM转化乳酸菌口服液的制备
添加有浓度为20μg/ml的红霉素的MRS液体培养基蒸汽灭菌，取经PCR验证及诱导表达实验鉴定的基因稳定转化的乳酸菌(实施例5制备)接种，30℃静置培养至OD600值达0.8～1.0时，离心，弃上清，取菌体沉淀加浓度为2％(w/v)的L-阿拉伯糖溶液或20％的低聚果糖溶液重悬细菌，配制成每毫升不低于10 10活菌的新鲜菌液，分装后于4℃冰箱冷藏保存。
2、突变体GM转化乳酸菌冻干粉的制备
同实施例3。
3、突变体GM转化乳酸菌干燥粉剂的制备
同实施例3。
4、突变体GM转化大肠杆菌Nissle1917颗粒剂的制备
同实施例3。
5、突变体GM转化大肠杆菌Nissle1917片剂的制备
同实施例3。
6、突变体GM转化大肠杆菌Nissle1917胶囊剂的制备
同实施例3。
以下通过试验例的方式来证明本发明的有益效果：
实验例1本发明突变体的重组乳酸菌表达及活性验证
1、本发明GLP-1突变体的乳酸菌表达
(1)将GLP-1GM，即SEQ ID NO：2所示的核苷酸序列构建到乳酸菌表达载体pMG36e质粒上SalI和HindIII位点，电转化乳酸杆菌后，挑取重组乳酸杆菌的单菌落，培养后提取质粒，PCR检验和测序检测GLP-1GM基因存在于该受体菌中。
(2)将上述的重组乳酸菌接种到添加有浓度为20μg/ml的红霉素的MRS培养基上，30℃静置培养至OD 600值达到0.8～1.0时，离心，弃上清，取菌体，备用。
2、GLP-1突变体的活性验证
按照每只大鼠灌胃1ml重组乳酸菌OD 600＝1.2，处理STZ诱导的T2DM动物模型，每组6只大鼠，在灌胃后各时间点，断尾取血，采用三诺血糖仪(GA-3型)测定一次血糖，用以确定口服重组乳酸菌-GLP-1GM突变体的降糖效果。
结果如图4所示。图4中，26小时后的降糖效果为24h补喂重组乳酸菌后2h的降糖效果。
可见，重组乳酸菌中表达的GLP-1GM突变体有一定的降糖效果，与在大肠杆菌中的结果相似，GLP-1GM的半衰期仍然较短，灌胃2小时血糖浓度下降(P＜0.01)，5-7小时后血糖略有回升(P＜0.05)，24h降糖效果减弱，如果立即二次灌胃等量的重组乳酸菌，2h后(即26h)又获得降糖效应(P＜0.01)。
因此，重组乳酸菌中表达的GLP-1GM突变体同样具有口服降糖效果。
重组乳酸菌中表达的GLP-1GM突变体同样具有口服降糖效果。与天然的GLP-1相比，本发明GLP-1突变体，减小了DPP-Ⅳ的降解作用，半衰期约为3h；而且本发明构建的含有GLP-1突变体的重组益生菌具有持续降糖作用。
实验例2突变体GM转化大肠杆菌Nissle1917减肥作用研究
1、实验动物
SPF级昆明小鼠，体重20-25g，购自成都达硕实验动物有限公司。合格证号：SCXK(川)2015-030。动物接收后进行检疫及适应性观察3天，合格后 开始实验。动物自由饮水摄食，温度(20±2)℃，湿度(60±5)％，12h光照周期。
2、实验动物分组
实验动物随机分为：正常组、模型组、重组大肠杆菌Nissle1917(实施例5制备)高中低剂量组，每组10只动物。
3、实验动物造模及给药方法
正常组喂食普通饲料，其余四组喂食高脂高糖饲料(饲料配方为：普通饲料67％，蔗糖20％，猪油10％，胆固醇2％，胆酸钠1％)。试验动物每天自由进食及饮水。重组大肠杆菌Nissle1917高中低剂量组分别灌胃活菌数为10 10、10 9、10 8CFU菌液各0.3ml，模型组灌胃等体积灭菌生理盐水，每日一次，分别在第四周及第八周取材测定。
4、检测指标
(1)体重：每周固定时间测定实验动物体重，并计算体重增长值。
(2)摄食量：每周固定时间测定每组实验动物的给食量和剩食量，计算摄食量。
(3)空腹血糖及口服糖耐量(OGTT)测定：第八周，动物禁食12h后称取体重测定空腹血糖后，每只小鼠一次性灌胃浓度为50％的葡萄糖溶液3g/kg，灌胃葡萄糖后30min、60min、120min、180min尾尖取血采用三诺血糖仪(GA-3型)测定各时间点血糖值，并计算曲线下面积(AUC)。
(4)体内脂肪重量、脂体比及肝脏重量测定：实验结束后，处死动物，剥离肝脏、肾周脂肪组织和睾丸(卵巢)周围脂肪组织并称重。分别计算肾周脂肪组织和睾丸(卵巢)周围脂肪组织与体重的比值即脂体比。同时剥离肝脏，称取肝脏重量。
5、实验结果
(1)体重及体重增长值
如图5所示，模型组体重及体重增长值较正常组均显著升高(p＜0.05)。与模型组相比，四周时菌液高中低剂量组均可显著降低实验动物的体重及体重增长值(p＜0.05)，八周时菌液高中剂量组可显著降低实验动物的体重及体重增长值(p＜0.05)。
(2)摄食量
如图6所示，与模型组相比，菌液高中低剂量组均可显著降低实验动物的摄食量(p＜0.05)。与正常组相比，菌液高中低剂量组的摄食量无显著性差异(p＞0.05)。
(3)血糖和口服糖耐量
如图7所示，各组间空腹血糖无显著性差异(p＞0.05)。灌胃葡萄糖溶液 后，除正常组外，各组血糖均显著升高。计算各组血糖浓度AUC，与模型组相比，菌液高中剂量组均可显著降低实验动物糖耐量试验的AUC(p＜0.05)。
(4)体内脂肪质量及脂体比
如图8所示，模型组的肾脏周围脂肪组织及脂体比较正常组均显著升高(p＜0.05)。与模型组相比，第四周时，菌液高剂量组可显著降低实验动物的肾脏周围脂肪组织及脂体比(p＜0.05)；第八周时，菌液高中剂量组均可显著降低实验动物的肾脏周围脂肪组织及脂体比(p＜0.05)。
如图9所示，模型组的睾丸(卵巢)周围脂肪组织及脂体比较正常组均显著升高(p＜0.05)。与模型组相比，第四周时菌液高剂量组可显著降低实验动物的睾丸(卵巢)周围脂肪组织及脂体比(p＜0.05)；第八周时菌液高中剂量组均可显著降低实验动物的睾丸(卵巢)周围脂肪组织及脂体比(p＜0.05)。
(5)肝脏重量
如图10所示，模型组的肝脏组织重量比较正常组均显著升高(p＜0.05)。与模型组相比，菌液高剂量组可显著降低实验动物的肝脏重量(p＜0.05)。
上述结果表明，突变体GM转化大肠杆菌Nissle1917可抑制实验小鼠的食欲，减少能量吸收；显著降低实验小鼠的体重、体内脂肪含量、脂体比和肝脏的重量，具有十分明显的减肥降脂的作用。同时该转化大肠杆菌Nissle1917还可显著降低口服糖耐量试验中血糖变化的曲线下面积，具有一定的降低血糖的趋势。

Claims (23)

  1. 一种GLP-1突变体，其特征在于：它是在野生型GLP-1基础上，经过氨基酸位点突变得到，它的氨基酸序列如SEQ ID NO：1所示。
  2. 编码SEQ ID NO：1所示氨基酸序列的核苷酸序列。
  3. 根据权利要求2所述的核苷酸序列，其特征在于：序列如SEQ ID NO：2所示。
  4. 一种重组载体，其特征在于：它包含权利要求2或3所示的核苷酸序列；进一步地，所述重组载体是重组原核载体；更进一步地，所述原核载体为pGEX质粒或者pMG36e载体。
  5. 一种重组菌，其特征在于：它包含权利要求4所述的重组载体；进一步地，所述重组菌为重组大肠杆菌Nissle 1917或者重组乳酸菌。
  6. 一种制备权利要求1或2所述GLP-1突变体的方法，其特征在于：包含如下步骤：
    取权利要求5所述的重组大肠杆菌Nissle 1917，接种到LB培养基上，振荡培养至OD 600为0.8-1.0时，离心，取沉淀，分离纯化，即可。
  7. 根据权利要求6所述的方法，其特征在于：当重组菌为大肠杆菌Nissle1917时，培养基为LB培养基。
  8. 根据权利要求6所述的方法，其特征在于：重组菌为乳酸菌时，培养基为MRS培养基。
  9. 权利要求1或2所述突变体、权利要求3所述核苷酸序列、权利要求4所述重组载体、权利要求5所述重组菌在制备治疗糖尿病的药物中的用途。
  10. 一种治疗糖尿病的药物，其特征在于：它含有权利要求1所述突变体、权利要求2或3所述核苷酸序列、权利要求4所述重组载体和/或权利要求5所述重组菌。
  11. 权利要求1或2所述突变体、权利要求3所述核苷酸序列、权利要求4所述重组载体、权利要求5所述重组菌在制备治疗肥胖的药物中的用途。
  12. 一种减肥的药物，其特征在于：它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分。
  13. 一种减肥的保健食品，其特征在于：它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分。
  14. 一种用于治疗糖尿病或者减肥的药物，其特征在于：它以氨基酸序列如SEQ ID NO：1所示GLP-1突变体、SEQ ID NO：2所示核苷酸序列、包含SEQ ID NO：2所示的核苷酸序列的重组载体和/或包含SEQ ID NO：2所示核苷酸序列的重组菌为有效成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂；所述制剂为口服制剂。
  15. 根据权利要求14所述的药物，其特征在于：所述口服制剂为片剂、胶囊剂、粉剂、颗粒剂或口服液。
  16. 根据权利要求15所述的药物，其特征在于：所述粉剂为冻干粉剂。
  17. 根据权利要求16所述的药物，其特征在于：所述冻干粉按照如下方法制备：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，分装后冻干，即可；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
  18. 根据权利要求15所述的药物，其特征在于：所述粉剂为干燥粉剂，所述干燥粉剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，烘干，烘干的温度不超过40℃；所述混合液体中，低聚果糖溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)，优选为2：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
  19. 根据权利要求15所述的药物，其特征在于：所述制剂为颗粒剂，所述颗粒剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，制粒，得颗粒剂；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
  20. 根据权利要求15所述的药物，其特征在于：所述制剂为片剂，所述片剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，压片，得片剂。
  21. 根据权利要求20所述得药物，其特征在于：所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
  22. 根据权利要求15所述的药物，其特征在于：所述制剂为胶囊剂，所述胶囊剂按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液重悬细菌，再加入浓度为15％(w/v)的甘油溶液、浓度为10％(w/v)的脱脂奶粉溶液、浓度为3％(w/v)的乳糖溶液和浓度为3％(w/v)的海藻糖溶液，配制成每克为10 10～10 12cfu活菌的混合液体，干燥，得粉剂，加入0.5～2倍重量的矫味剂、0.5～2倍重量的硬脂酸镁，混合，制粒，得颗粒剂，装胶囊，得胶囊剂；所述混合液体中，低聚果糖溶液、甘油溶液、脱脂奶粉溶液、乳糖溶液与海藻糖溶液的体积比为(1～10)：(1～10)：(1～10)：(1～10)：(1～10)，优选为2：3：5：6：1；优选地，所述每克混合液体中的活菌数量为10 10cfu。
  23. 根据权利要求15所述的药物，其特征在于：所述制剂为口服液，所述口服液按照如下方法：取包含SEQ ID NO：2所示核苷酸序列的重组菌的细菌沉淀，加浓度为20％(w/v)的低聚果糖溶液或者浓度为2％(w/v)的L-阿拉伯糖溶液重悬细菌，配制成每毫升不低于10 10～10 12cfu活菌的新鲜菌液，灌装，即可；优选地，所述每克混合液体中的活菌数量为10 10cfu。
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