CN110251661B

CN110251661B - A medicinal preparation for treating diabetes or reducing weight

Info

Publication number: CN110251661B
Application number: CN201811520996.3A
Authority: CN
Inventors: 顾建文; 马婕; 马永平
Original assignee: Fuzhou Taijiang Xijiya Health Technology Co ltd
Current assignee: Fuzhou Taijiang Xijiya Health Technology Co ltd
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2022-09-16
Anticipated expiration: 2038-12-12
Also published as: CN110251661A

Abstract

The invention discloses a medicine for treating diabetes or losing weight, which is prepared from amino acid sequences shown as SEQ ID NO: 1, GLP-1 mutant shown in SEQ ID NO: 2, comprising the nucleotide sequence shown in SEQ ID NO: 2 and/or a recombinant vector comprising the nucleotide sequence shown in SEQ ID NO: 2 is a preparation prepared by taking the recombinant bacteria of the nucleotide sequence shown in the formula 2 as an effective component and adding pharmaceutically acceptable auxiliary materials or auxiliary components; the preparation is an oral preparation. The medicine containing the GLP-1 mutant can effectively reduce the blood sugar concentration or lose weight and reduce fat, can be used for treating type II diabetes or obesity, and has good clinical application prospect.

Description

A medicinal preparation for treating diabetes or reducing weight

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a medicament for treating diabetes or losing weight.

Background

With the change of dietary structure and lifestyle of people, obesity has become an epidemic worldwide. The incidence of overweight and obesity is currently on the increasing trend year by year around the world. In particular, the incidence of overweight and obesity in our country has approached or even reached the initial level in developed countries in recent years. For those with a high BMI index, obesity can cause an increase in the incidence of other diseases, most notably diabetes, cardiovascular disease and cancer, and overweight/obesity severely harms human health.

Meanwhile, overweight/obesity is an independent risk factor for the development and progression of Type 2Diabetes mellitis (T2DM), both of which are concomitant disease states. Studies show that every increase of body mass index is 1kg/m ² The risk of T2DM increased by 12%; whereas in patients with T2DM, the prevalence of overweight and obesity is more than 80%. At the same time, overweight and obese states can further exacerbate the insulin resistance state of patients with T2DM and greatly increase the risk of complications such as cardiovascular disease. Enhanced weight loss may bring definite benefits to T2DM patients: relieving insulin resistance, repairing impaired beta cell function of pancreatic islet, and optimizing blood glucoseControlling and improving the related risk factors of cardiovascular diseases, and the like. Obesity and the vicious circle state caused by T2DM make the traditional treatment mode taking 'sugar reduction' as the core go into a dilemma, which also makes more and more scholars pay attention to the importance of 'weight reduction' treatment.

Although the weight of the obese patient can be reduced by the simple lifestyle intervention, the long-term curative effect is not ideal, and on the one hand, the lifestyle intervention is related to the fact that most patients hardly adhere to the weight-reducing lifestyle strictly; on the other hand, the weight of the traditional Chinese medicine is inevitably increased during the application process.

However, many weight-reducing drugs gradually exit the market due to serious safety problems while bringing good weight-reducing efficacy. Currently, clinically applied weight-reducing medicines mainly comprise orlistat capsules approved in 1999, lorcaserin, phentermine and topiramate capsules approved in 2012, naltrexone hydrochloride and bupropion compound sustained-release tablets approved in 2014 and liraglutide injection.

Liraglutide (Liraglutide) is a glucagon-like peptide 1 (GLP-1) analog peptide, and is a novel drug based on the action of incretin. GLP-1 analog peptide mainly reduces the intake of energy of a patient by inhibiting central feeding desire, inhibiting gastrointestinal peristalsis and increasing satiety, thereby exerting the effect of losing weight. Linagliptin has glucose concentration dependent blood glucose lowering effect, and single drug therapy can not cause hypoglycemia.

Liraglutide was marketed in 2009 and 2010 in the united states and european union, respectively, for the treatment of type 2diabetes (T2 DM). In 2014 and 2015, FDA and European drug administration approve the GLP-1 analog peptide as a supplement for diet control and physical exercise for treating chronic obesity, the effect of the GLP-1 analog peptide is superior to that of orlistat which is a main diet-reducing drug on the market, the weight-reducing effect is clear, and the GLP-1 analog peptide is used for treating obesity for the first time of day. Liraglutide was approved for marketing in china in 2011. Long-term tests show that the liraglutide can effectively reduce the weight of patients, and is suitable for adults with a Body Mass Index (BMI) of more than or equal to 30 or adults with a BMI of 27 or more and at least one obesity complication such as T2DM, hypertension or high cholesterol. At present, the strong desire for weight loss of people and the existing weight-reducing medicines with poor curative effect form a big contradiction, the development of the weight-reducing medicines with GLP-1-like peptides is a good choice. Therefore, the medicine with the dual benefits of losing weight and reducing blood sugar has very wide market prospect.

At present, GLP-1 analog peptide polypeptide medicaments including liraglutide all need to be injected and administered, are directly and orally taken and are ineffective, and have the defects of inconvenient administration and the like. Treatment of diabetes, however, often requires long-term, continuous administration. The injection administration mode is inconvenient to use and carry, poor in medication compliance, and also has risks of irritation, anaphylactic reaction and the like, and great physiological and psychological pains are brought to patients. The oral preparation is most in line with the habit of taking medicine of people, and the preparation process is mature and simple. Therefore, starting from changing the administration route of the GLP-1 similar peptide, the GLP-1 similar peptide is convenient to develop and carry, is suitable for GLP-1 similar peptide medicaments for long-term use, and has great significance.

Disclosure of Invention

The object of the present invention is to provide a medicament comprising a novel GLP-1 mutant.

The invention provides a medicine for treating diabetes or losing weight, which is prepared from amino acid sequences shown as SEQ ID NO: 1, and the GLP-1 mutant shown in SEQ ID NO: 2, comprising the nucleotide sequence shown in SEQ ID NO: 2 and/or a recombinant vector comprising the nucleotide sequence shown in SEQ ID NO: 2 is a preparation prepared by taking the recombinant bacteria of the nucleotide sequence shown in the formula 2 as an effective component and adding pharmaceutically acceptable auxiliary materials or auxiliary components; the preparation is an oral preparation.

Preferably, the oral preparation is a tablet, a capsule, a powder, a granule or an oral liquid.

Preferably, the powder is a lyophilized powder.

Further, the freeze-dried powder is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) for suspending the bacteria, and then adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v) and a skim milk powder solution with the concentration of 3% (w/v) for suspending the bacteriaLactose solution and trehalose solution with concentration of 3% (w/v) are prepared into 10 g/g ¹⁰ ～10 ¹² Subpackaging the mixed liquid of the live bacteria cfu, and freeze-drying to obtain the compound preparation; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1 to 10), preferably 2: 3: 5: 6: 1; preferably, the number of the viable bacteria per gram of the mixed liquid is 10 ¹⁰ cfu。

Preferably, the powder is a dry powder, which is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 per gram ¹⁰ ～10 ¹¹ Drying the mixed liquid of the cfu viable bacteria at the temperature not more than 40 ℃; in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the skim milk powder solution, the lactose solution and the trehalose solution is (1-10): (1-10): (1-10): (1-10), preferably 2: 5: 6: 1; preferably, the number of the viable bacteria per gram of the mixed liquid is 10 ¹⁰ cfu。

Preferably, the formulation is a granule, which is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Drying the mixed liquid of the cfu viable bacteria to obtain powder, adding 0.5-2 times of flavoring agent and 0.5-2 times of magnesium stearate by weight, mixing, and granulating to obtain granules; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10), preferably 2: 3: 5: 6: 1; preferably, said per gram is mixedThe number of viable bacteria in the liquid is 10 ¹⁰ cfu。

Preferably, the formulation is a tablet, which is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² And drying the mixed liquid of the cfu viable bacteria to obtain powder, adding 0.5-2 times of flavoring agent and 0.5-2 times of magnesium stearate by weight, mixing, and tabletting to obtain the tablet.

Preferably, in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the glycerol solution, the skim milk powder solution, the lactose solution and the trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10), preferably 2: 3: 5: 6: 1; preferably, the number of the viable bacteria per gram of the mixed liquid is 10 ¹⁰ cfu。

Preferably, the preparation is a capsule, and the capsule is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Drying the mixed liquid of the cfu viable bacteria to obtain powder, adding 0.5-2 times of flavoring agent and 0.5-2 times of magnesium stearate by weight, mixing, granulating to obtain granules, and encapsulating to obtain capsules; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10), preferably 2: 3: 5: 6: 1; preferably, the number of the viable bacteria per gram of the mixed liquid is 10 ¹⁰ cfu。

Preferably, the preparation is an oral liquid, and the oral liquid is prepared by the following steps: taking a peptide comprising SEQ ID NO: 2 coreBacterial precipitation of recombinant bacteria of nucleotide sequence, adding fructo-oligosaccharide solution with concentration of 20% (w/v) or L-arabinose solution with concentration of 2% (w/v) for resuspending bacteria, and preparing into bacteria with concentration of not less than 10 per ml ¹⁰ ～10 ¹² Filling fresh bacterial liquid of live cfu bacteria; preferably, the number of the viable bacteria per gram of the mixed liquid is 10 ¹⁰ cfu。

Compared with natural GLP-1, the GLP-1 mutant reduces the degradation of DPP-IV and has half-life of about 3 h; moreover, the recombinant probiotics containing the GLP-1 mutant constructed by the invention can be prepared into various solid and liquid preparations to realize oral administration, so that the pain of patients in long-term injection is avoided; meanwhile, after being taken by a human body, the genetically engineered probiotics can survive and colonize in intestinal tracts of the human body to become a functional in-vivo bioreactor, and GLP-1 mutant polypeptide is continuously generated and secreted, so that the effects of continuously reducing blood sugar and treating obesity are achieved, and the clinical application prospect is good.

It will be apparent that various other modifications, substitutions and alterations can be made in the present invention without departing from the basic technical concept of the invention as described above, according to the common technical knowledge and common practice in the field.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 results of screening GLP-1GM mutant proteins of the invention.

FIG. 2 shows the results of SDS-PAGE and WB detection of GLP-1GM mutants of the present invention.

FIG. 3 the hypoglycemic effect of the Escherichia coli expressed GLP-1GM mutant gavage T2DM rat model of the present invention.

FIG. 4 shows the hypoglycemic effect of the Lactobacillus-expressing GLP-1GM mutant gavage T2DM rat model.

FIG. 5 effect of mutant GM transformed E.coli Nissle1917 on body weight and body weight gain values of mice, Δ p < 0.05, Δ Δ p < 0.01 compared to normal group; p < 0.05, p < 0.01 compared to model group.

FIG. 6 effect of mutant GM transformed E.coli Nissle1917 on food intake of mice, Δ p < 0.05 and Δ Δ p < 0.01 compared to normal group; p < 0.05, p < 0.01 compared to model group.

FIG. 7 shows the effect of the mutant GM transformed Escherichia coli Nissle1917 on the fasting blood glucose and the area under the blood glucose concentration change curve of the mice, wherein compared with the normal group, the delta p is less than 0.05, and the delta p is less than 0.01; p < 0.05, p < 0.01 compared to model group.

FIG. 8 shows the effect of mutant GM transformed E.coli Nissle1917 on the weight and lipid-to-body ratio of the adipose tissues around the kidney of mice, compared with normal group, Δ p is less than 0.05, Δ Δ p is less than 0.01; p < 0.05, p < 0.01 compared to model group.

FIG. 9 the effect of mutant GM transformed E.coli Nissle1917 on the weight and lipid-to-body ratio of adipose tissue surrounding mouse testis (ovary), Δ p < 0.05 and Δ Δ p < 0.01 compared to normal group; p < 0.05, p < 0.01 compared to model group.

FIG. 10 effect of mutant GM transformed E.coli Nissle1917 on mouse liver weight, Δ p < 0.05, Δ Δ p < 0.01 compared to normal group; p < 0.05, p < 0.01 compared to model group.

Detailed Description

The following examples are further illustrative, but the present invention is not limited to these examples.

The experimental reagents and instruments used in the invention are as follows:

strains and plasmids such as pGEX-4T-1, pMG36e, pET32a, E.coli TOP10, E.coli BL21(DE3), Escherichia coli Nissle1917, lactobacillus, lactococcus lactis and the like are stored in the biochemistry and molecular biology research laboratory of Chongqing medical university from Hill Biotech, Inc. and Hill Ci Biotechnology; male rats were provided by the experimental animals center of Chongqing university of medicine. T4 ligase, Taq common enzyme, Sal I, BamH I, protein Marker, DNA Marker, Plasmid Mini Kit I, Cycle-Pure Kit, gel recovery Kit, IPTG, erythromycin, ampicillin, zotocin, BCA protein concentration determination Kit, PMSF and other reagents (all of which are commercially available).

Example 1 screening and Activity detection of mutant sequences of the present invention

Starting from an original GLP-1 sequence, 4 mutant polypeptide sequences are reconstructed and designed, and a polypeptide product with the purity of up to 85 percent is obtained through chemical synthesis (medium peptide biochemistry). The polypeptide sequence is as follows:

GLP-1 fragment (7-37 aa): HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG

GM(SEQ ID NO：1)：

HHEGTFTSDVSSYLEGQAAKKFIAWLVRGGKKKKKYGRKKRRQRRREF

C33：HACGTFTSDVSSYLEGQAAKEFIAWLCKGRG

K34：HAEGTFTSDVSSYLEGQAAKEFIAWLVKKKK

R34：HAEGTFTSDVSSYLEGQAAKEFIAWLVRRRR

The GM is obtained by mutating alanine at position 8 of GLP-1 into histidine, mutating glutamic acid at position 27 into lysine, mutating lysine at position 34 into arginine, mutating arginine at position 36 into glycine, mutating glycine at position 37 into lysine on the basis of GLP-1 original sequence, and adding a segment of sequence at the tail end.

C33 is obtained by mutating the 9 th glutamic acid and 33 th valine of GLP-1 to cysteine on the basis of GLP-1 original sequence.

K34 is obtained by mutating 35 th glycine, 36 th arginine and 37 th glycine of GLP-1 to lysine on the basis of GLP-1 original sequence.

R34 is obtained by mutating 34 th lysine to arginine, 35 th glycine to arginine and 37 th glycine to arginine of GLP-1 based on GLP-1 original sequence.

Differences between the original sequence of GLP-1 and mutants R34, K34 and C33 are shown in Table 1.

TABLE 1 sequence comparison of GLP-1 and mutants

Male rats, 6 per group, were fasted for 12h, and after measurement of blood glucose, 20mmol/kg glucose was intraperitoneally injected and 50. mu.g/kg of the above four mutant polypeptides was administered intraperitoneally. Blood glucose was measured with a Sano blood glucose meter (GA-3 type) at each time point after the administration.

The results are shown in FIG. 1.

The natural GLP-1 is easily and rapidly hydrolyzed and inactivated by dipeptidyl peptidase IV (DPP-IV), the half-life period is less than 5min, and the natural GLP-1 is not suitable for clinical application, so the animal experiment is not carried out.

As can be seen, the GLP-1 mutant (GLP-1GM) with the number of GM has better hypoglycemic activity and a half-life period of 3 hours, while other GLP-1 mutants have poorer hypoglycemic capability and half-life period.

Example 2 recombinant E.coli expression and Activity verification of the mutants of the invention

1. Escherichia coli expression of GLP-1 mutant of the present invention

(1) Adding a signal peptide sequence in front of the sequence of the GLP-1 mutant GM, adding an HIV cell-penetrating peptide sequence and 6 His-tag sequences at the 3' -end to construct a GLP-1 mutant capable of secreting and expressing and penetrating cell membranes, wherein the nucleotide sequence of the GLP-1 mutant is shown as SEQ ID NO: 2, respectively.

SEQ ID NO：2：

atgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgctggatcccaccacgagggcaccttcacctccgacgtgtcctcctacctggagggccaggccgccaagaagttcatcgcctggctggtgcgcggcggcaagaagaagaagaagtacggccgcaagaagcgccgccagcgccgccgcctcgaggacgacgacgacaagcaccatcaccatcaccattaa

(2) Converting SEQ ID NO: 3 to BamHI and SalI sites of an Escherichia coli expression vector pGEX to obtain a pGEX-GLP-1GM vector, and after the sequencing is correct, the Escherichia coli E.coli BL21(DE3) is transformed, and the size of a target fragment is about 12 KD. The expression result is identified to be correct by Western blot after being purified by nickel-containing magnetic beads, and the result is shown in figure 2. As can be seen, the GLP-1 mutant protein is successfully expressed by the invention.

(3) pGEX-GLP-1GM expression vector is transformed into Escherichia coli Nissle1917, and the success of pGEX-GLP-1GM transformation is identified through PCR and sequencing. Taking recombinant Escherichia coli Nissle1917, inoculating to LB medium added with ampicillin with final concentration of 100 μ g/ml, shake culturing at 37 deg.C and 250r/min to OD ₆₀₀ The value reaches 0.8-1.0, the bacterial liquid is centrifuged, the supernatant is discarded, and the OD of the precipitate is adjusted by PBS ₆₀₀ The value is 1.2.

3. Activity verification of GLP-1 mutant

The recombinant Escherichia coli (OD) was orally administered to rats in an oral gavage of 1ml per rat ₆₀₀ 1.2) treatment of the STZ induced a successful T2DM rat model, 6 rats per group, at various time points after gavage, blood was taken by tail-biting and blood glucose was measured once using a trino glucometer (GA-3 type) to determine the hypoglycemic effect of the oral GLP-1GM mutant. The results are shown in FIG. 3.

It can be seen that GLP-1GM mutant expressed in E.coli Nissle1917 has hypoglycemic effect 2 hours after intragastric administration (P < 0.01 compared with the control group without intragastric administration). After 8 hours of gastric lavage, the blood sugar slightly rises.

EXAMPLE 3 preparation of mutant GM transformed E.coli Nissle1917

1. Preparation of mutant GM transformed Escherichia coli Nissle1917 oral liquid

Adding erythromycin resistance LB liquid culture medium with concentration of 200 μ g/ml for steam sterilization, inoculating Escherichia coli Nissle1917 (prepared in example 2) with stable gene conversion identified by PCR verification and induction expression experiment, shake culturing at 37 deg.C and 250r/min until OD600 value reaches 0.8-1.0, centrifuging for 5min to harvest bacteria, washing with normal saline for 2 times, centrifuging to collect thallus precipitate, adding 2% (w/v) L-arabinose solution or 20% fructo-oligosaccharide solution for resuspension, and making into 10 ml of bacteria ¹⁰ And (4) filling fresh bacterial liquid of the live cfu bacteria, and refrigerating and storing in a refrigerator at 4 ℃.

2. Preparation of mutant GM transformed Escherichia coli Nissle 1917/lyophilized powder

Taking the bacterial precipitate obtained by the method, adding fructo-oligosaccharide with the concentration of 20% (w/v) for resuspending bacteria, and adding glycerol with the concentration of 15% (w/v)Oil, 10% (w/v) skimmed milk powder, 3% (w/v) lactose and 3% (w/v) trehalose formulated to give 10 g/g ¹⁰ The mixed liquid of the cfu viable bacteria comprises a fructo-oligosaccharide solution of 20% (w/v), a glycerol solution of 15% (w/v), a skim milk powder solution of 10% (w/v), a lactose solution of 3% and a trehalose solution of 3% in a volume ratio of 2: 3: 5: 6: 1, subpackaging, freeze-drying in a freeze dryer, sealing, and storing in a refrigerator at 4-8 ℃.

3. Preparation of mutant GM transformed Escherichia coli Nissle1917 dry powder

Taking the bacterial sediment harvested by the method, adding fructo-oligosaccharide with the concentration of 20% (w/v) to resuspend the bacteria, adding skimmed milk powder with the concentration of 10% (w/v), lactose with the concentration of 3% (w/v) and trehalose with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ The mixed liquid of cfu viable bacteria comprises a fructo-oligosaccharide solution of 20% (w/v), a skim milk powder solution of 10% (w/v), a lactose solution of 3% and a trehalose solution of 3% in a volume ratio of 2: 5: 6: 1, subpackaging, placing on a vacuum drying instrument, drying at the highest temperature of not more than 40 ℃, sealing, and storing in a refrigerator at 4-8 ℃.

4. Preparation of mutant GM transformed Escherichia coli Nissle1917 granules

And adding 0.5-2 parts of flavoring agent into the prepared transformed escherichia coli Nissle1917 freeze-dried powder/dried powder, uniformly mixing the flavoring agent and the sweetening agent for 20-30min, putting the mixture into a dry granulating machine for granulation, collecting particles of 10-100 meshes, and subpackaging to obtain transformed escherichia coli Nissle1917 granules.

5. Preparation of mutant GM transformed E.coli Nissle1917 tablets

And (3) putting the prepared escherichia coli Nissle1917 freeze-dried powder, 0.5-2 parts of flavoring agent and 0.5-2 parts of magnesium stearate into a powder mixer, mixing for 5-15 min, uniformly mixing to obtain a raw material capable of being directly tabletted, and putting the raw material into a single-punch tabletting machine for tabletting to obtain the transformed escherichia coli Nissle1917 tablets.

6. Preparation of capsules of mutant GM transformed Escherichia coli Nissle1917

And filling the prepared escherichia coli Nissle1917 granules into hollow hard capsules according to corresponding specifications, and putting the obtained capsules into a polishing machine to remove redundant dust to obtain the transformed escherichia coli Nissle1917 capsules.

Example 4 transformation of lactic acid bacteria with mutant GM

1. Transformation of mutant GM into lactic acid bacteria

(1) GLP-1GM, SEQ ID NO: 2 to SalI and HindIII sites on a plasmid of a lactobacillus expression vector pMG36e, electrically transforming lactobacillus, selecting a single colony of recombinant lactobacillus, culturing, extracting the plasmid, and detecting the existence of the GLP-1GM gene in the recipient bacterium by PCR (polymerase chain reaction) inspection and sequencing.

(2) Inoculating the recombinant lactobacillus to MRS culture medium containing erythromycin with concentration of 20 μ g/ml, and standing at 30 deg.C for culture to OD ₆₀₀ When the value reaches 0.8-1.0, centrifuging, discarding the supernatant, and taking the thalli for later use.

Example 5 preparation of a preparation of mutant GM transformed lactic acid bacteria

1. Preparation of mutant GM transformed lactobacillus oral liquid

Adding erythromycin with concentration of 20 μ g/ml into MRS liquid culture medium, steam sterilizing, inoculating lactic acid bacteria (prepared in example 5) with stable gene conversion identified by PCR verification and induction expression experiment, standing at 30 deg.C, culturing until OD600 value reaches 0.8-1.0, centrifuging, discarding supernatant, adding 2% (w/v) L-arabinose solution or 20% fructo-oligosaccharide solution into thallus precipitate, and resuspending bacteria to obtain a suspension containing no less than 10 ml of bacteria ¹⁰ And (4) subpackaging fresh bacteria liquid of live bacteria, and refrigerating and storing in a refrigerator at 4 ℃.

2. Preparation of mutant GM transformed lactobacillus freeze-dried powder

The same as in example 3.

3. Preparation of mutant GM transformed lactobacillus dry powder

The same as in example 3.

4. Preparation of mutant GM transformed Escherichia coli Nissle1917 granules

The same as in example 3.

5. Preparation of mutant GM transformed E.coli Nissle1917 tablets

The same as in example 3.

6. Preparation of capsules of mutant GM transformed Escherichia coli Nissle1917

The same as in example 3.

The beneficial effects of the invention are demonstrated by means of test examples as follows:

experimental example 1 recombinant lactic acid bacteria expression and Activity verification of the mutant of the present invention

1. Lactobacillus expression of GLP-1 mutants of the invention

2. Activity verification of GLP-1 mutant

The recombinant lactobacillus OD is filled into the stomach of each rat in an amount of 1ml ₆₀₀ STZ-induced T2DM animal models were treated as 1.2, 6 rats per group, and at various time points after gavage, blood was removed by tail-biting and blood glucose was measured once using a trino glucometer (type GA-3) to determine the hypoglycemic effect of the oral recombinant lactic acid bacteria-GLP-1 GM mutant.

The results are shown in FIG. 4. In fig. 4, the effect of lowering blood sugar after 26 hours is the effect of lowering blood sugar 2 hours after 24 hours of feeding recombinant lactic acid bacteria.

It can be seen that the GLP-1GM mutant expressed in the recombinant lactobacillus has a certain blood sugar reduction effect, the half-life period of the GLP-1GM is still shorter, the blood sugar concentration is reduced after 2 hours of gastric lavage (P is less than 0.01), the blood sugar is slightly increased after 5-7 hours (P is less than 0.05), the blood sugar reduction effect is weakened after 24 hours, and if the recombinant lactobacillus with the same amount is subjected to secondary gastric lavage immediately, the blood sugar reduction effect is obtained after 2 hours (namely 26 hours) (P is less than 0.01).

Therefore, the GLP-1GM mutant expressed in the recombinant lactic acid bacteria also has the oral hypoglycemic effect.

Experimental example 2 study on the weight-reducing effect of mutant GM transformed into Escherichia coli Nissle1917

1. Laboratory animal

SPF-grade Kunming mice, weighing 20-25g, were purchased from WUDUDO laboratory animals, Inc. Certificate number: SCXK (Chuan) 2015-030. Animals were received and quarantined and observed for 3 days for testing after qualification. Animals are freely drunk with water, the temperature is 20 +/-2 ℃, the humidity is 60 +/-5 percent, and the illumination period is 12 hours.

2. Grouping of experimental animals

Experimental animals were randomly divided into: normal group, model group, recombinant e.coli Nissle1917 (prepared in example 5) high, medium and low dose group, 10 animals per group.

3. Model making and administration method for experimental animal

The normal group is fed with common feed, and the other four groups are fed with high-fat high-sugar feed (the feed formula comprises 67% of common feed, 20% of cane sugar, 10% of lard, 2% of cholesterol and 1% of sodium cholate). The test animals had free access to food and water daily. The number of live intragastric bacteria in the recombinant escherichia coli Nissle1917 high-medium-low dose groups is 10 ¹⁰ 、10 ⁹ 、10 ⁸ 0.3ml of CFU bacterial liquid respectively, and the model group is perfused with sterilized normal saline with the same volume as the stomach once a day, and the materials are respectively taken and measured in the fourth week and the eighth week.

4. Detecting the index

(1) Weight: the body weight of the experimental animals was measured at fixed times per week and the body weight gain was calculated.

(2) Food intake: food intake and food remaining were measured for each group of experimental animals at fixed times per week and food intake was calculated.

(3) Fasting glucose and oral glucose tolerance (OGTT) assay: and in the eighth week, after animals are fasted for 12 hours, weighing the weight of the animals to measure the fasting blood glucose, measuring the blood glucose value at each time point by adopting a Sanuo glucometer (GA-3 type) after each mouse is fed with glucose solution with the gastric lavage concentration of 50% at one time by 3g/kg, and measuring the blood glucose value at each time point by adopting a Sanuo glucometer (GA-3 type) after the gastric lavage of the glucose solution for 30min, 60min, 120min and 180min, and calculating the area under the curve (AUC).

(4) In vivo fat weight, lipid ratio and liver weight determination: after the experiment, the animals were sacrificed and the liver, perirenal adipose tissue and peritesticular (ovarian) adipose tissue were stripped and weighed. The ratio of perirenal adipose tissue and peritesticular (ovarian) adipose tissue to body weight, i.e., the lipid body ratio, was calculated, respectively. The liver was also peeled off and weighed.

5. Results of the experiment

(1) Body weight and body weight gain

As shown in FIG. 5, the body weight and body weight gain of the model group were significantly higher than those of the normal group (p < 0.05). Compared with the model group, the bacterial liquid high, medium and low dose group can obviously reduce the weight and the weight growth value (p is less than 0.05) of the experimental animal at four weeks, and the bacterial liquid high, medium and high dose group can obviously reduce the weight and the weight growth value (p is less than 0.05) of the experimental animal at eight weeks.

(2) Food intake

As shown in FIG. 6, the feed intake of experimental animals was significantly reduced in the group with high, medium and low doses of the bacterial suspension compared to the model group (p < 0.05). Compared with the normal group, the food intake of the bacteria liquid high, medium and low dose group has no significant difference (p is more than 0.05).

(3) Blood glucose and oral glucose tolerance

As shown in FIG. 7, there was no significant difference in fasting plasma glucose between groups (p > 0.05). After the gastric perfusion of the glucose solution, the blood sugar of each group is obviously increased except the normal group. And calculating the blood glucose concentration AUC of each group, and compared with the model group, the bacterium liquid high and medium dose group can obviously reduce the AUC (p is less than 0.05) of the glucose tolerance test of the experimental animal.

(4) Body fat mass and body fat ratio

As shown in FIG. 8, the adipose tissues and lipids around the kidney were significantly increased in the model group compared to the normal group (p < 0.05). Compared with the model group, the bacterial liquid high-dose group can obviously reduce the fat tissue and the fat-body ratio (p is less than 0.05) around the kidney of the experimental animal at the fourth week; at the eighth week, the bacterial liquid high and medium dose groups can obviously reduce the fat tissues and the fat-body ratio (p is less than 0.05) around the kidney of the experimental animal.

As shown in FIG. 9, the adipose tissues and lipids around the testis (ovary) in the model group were significantly increased compared to the normal group (p < 0.05). Compared with the model group, the bacterial liquid high-dose group at the fourth week can obviously reduce the fat tissue and the fat-body ratio (p is less than 0.05) around the testis (ovary) of the experimental animal; the bacterial liquid high and medium dose group can obviously reduce the fat tissue and the fat-body ratio (p is less than 0.05) around the testis (ovary) of the experimental animal at the eighth week.

(5) Weight of liver

As shown in FIG. 10, the liver tissue weight was significantly increased in the model group compared to the normal group (p < 0.05). Compared with the model group, the bacterial liquid high-dose group can obviously reduce the weight of the liver of the experimental animal (p is less than 0.05).

The results show that the mutant GM transformed Escherichia coli Nissle1917 can inhibit the appetite of experimental mice and reduce energy absorption; obviously reduces the weight, the fat content in the body, the fat-to-body ratio and the weight of the liver of an experimental mouse, and has very obvious functions of losing weight and reducing fat. Meanwhile, the transformed escherichia coli Nissle1917 can also obviously reduce the area under the curve of blood glucose change in an oral glucose tolerance test, and has a certain tendency of reducing blood glucose.

SEQUENCE LISTING

<110> Sichuan Litong scientific biomedical science and technology Co., Ltd

<120> a pharmaceutical preparation for treating diabetes or losing weight

<130> GY848-18P1765

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 48

<212> PRT

<213> Artificial Sequence

<220>

<223> GM

<400> 1

His His Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Lys Phe Ile Ala Trp Leu Val Arg Gly Gly Lys Lys

20 25 30

Lys Lys Lys Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe

35 40 45

<210> 2

<211> 255

<212> DNA

<213> Artificial Sequence

<220>

<223> GLP-1 mutant GM

<400> 2

atgaaaaaga acatcgcatt cctcctggca tctatgtttg ttttctctat cgctaccaac 60

gcttacgctg gatcccacca cgagggcacc ttcacctccg acgtgtcctc ctacctggag 120

ggccaggccg ccaagaagtt catcgcctgg ctggtgcgcg gcggcaagaa gaagaagaag 180

tacggccgca agaagcgccg ccagcgccgc cgcctcgagg acgacgacga caagcaccat 240

caccatcacc attaa 255

<210> 3

<211> 31

<212> PRT

<213> GLP-1 fragments

<400> 3

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly

20 25 30

<210> 4

<211> 31

<212> PRT

<213> C33

<400> 4

His Ala Cys Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Cys Lys Gly Arg Gly

20 25 30

<210> 5

<211> 31

<212> PRT

<213> K34

<400> 5

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Lys Lys Lys

20 25 30

<210> 6

<211> 31

<212> PRT

<213> R34

<400> 6

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly

1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Arg Arg Arg

20 25 30

Claims

1. A medicament for treating diabetes or reducing weight, characterized by: the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1 and/or a mutant GLP-1 comprising SEQ ID NO: 2 is a preparation prepared by taking the recombinant bacteria of the nucleotide sequence shown in the formula 2 as an effective component and adding pharmaceutically acceptable auxiliary materials or auxiliary components; the preparation is an oral preparation.

2. The medicament of claim 1, wherein: the oral preparation is tablets, capsules, powder, granules or oral liquid.

3. The medicament of claim 2, wherein: the powder is freeze-dried powder.

4. The medicament of claim 3, wherein: the freeze-dried powder is prepared according to the following method: taking a polypeptide containing SEQID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Subpackaging the mixed liquid of the live bacteria cfu, and freeze-drying to obtain the compound preparation; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10).

5. The medicament of claim 4, wherein: in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the glycerol solution, the skim milk powder solution, the lactose solution and the trehalose solution is 2: 3: 5: 6: 1; the number of viable bacteria in the mixed liquid is 10 per gram ¹⁰ cfu。

6. The medicament of claim 2, wherein: the powder is dry powder, and the dry powder is prepared by the following steps: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) for resuspending bacteria, adding a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Drying the mixed liquid of the cfu viable bacteria at the temperature not more than 40 ℃; in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the skim milk powder solution, the lactose solution and the trehalose solution is (1-10): (1-10): (1-10): (1-10).

7. The medicament of claim 6, wherein: in the mixed liquid, lowerThe volume ratio of the polyfructose solution to the skim milk powder solution to the lactose solution to the trehalose solution is 2: 5: 6: 1; the number of viable bacteria in the mixed liquid is 10 per gram ¹⁰ cfu。

8. The medicament of claim 2, wherein: the preparation is granules, and the granules are prepared by the following steps: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Drying the mixed liquid of the cfu viable bacteria to obtain powder, adding 0.5-2 times of flavoring agent and 0.5-2 times of magnesium stearate by weight, mixing, and granulating to obtain granules; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10).

9. The medicament of claim 8, wherein: in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the glycerol solution, the skim milk powder solution, the lactose solution and the trehalose solution is 2: 3: 5: 6: 1; the number of viable bacteria in the mixed liquid is 10 per gram ¹⁰ cfu。

10. The medicament of claim 2, wherein: the preparation is a tablet, and the tablet is prepared according to the following method: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Mixing liquid of cfu viable bacteria, drying to obtain powder, adding 0.5-2 times of correctant and 0.5-EMagnesium stearate 2 times the weight of the tablet is mixed and tabletted to obtain the tablet.

11. The medicament of claim 10, wherein: in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10).

12. The medicament of claim 11, wherein: in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the glycerol solution, the skim milk powder solution, the lactose solution and the trehalose solution is 2: 3: 5: 6: 1; the number of viable bacteria in the mixed liquid is 10 per gram ¹⁰ cfu。

13. The medicament of claim 2, wherein: the preparation is a capsule, and the capsule is prepared by the following steps: taking a peptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) to resuspend the bacteria, adding a glycerol solution with the concentration of 15% (w/v), a skim milk powder solution with the concentration of 10% (w/v), a lactose solution with the concentration of 3% (w/v) and a trehalose solution with the concentration of 3% (w/v), and preparing into 10 g/g ¹⁰ ～10 ¹² Drying the mixed liquid of the cfu viable bacteria to obtain powder, adding 0.5-2 times of flavoring agent and 0.5-2 times of magnesium stearate by weight, mixing, granulating to obtain granules, and encapsulating to obtain capsules; in the mixed liquid, the volume ratio of a fructo-oligosaccharide solution, a glycerol solution, a skim milk powder solution, a lactose solution to a trehalose solution is (1-10): (1-10): (1-10): (1-10): (1-10).

14. The medicament of claim 13, wherein: in the mixed liquid, the volume ratio of the fructo-oligosaccharide solution, the glycerol solution, the skim milk powder solution, the lactose solution and the trehalose solution is 2: 3: 5: 6: 1; the number of viable bacteria in the mixed liquid is 10 per gram ¹⁰ cfu。

15. The medicament of claim 2, wherein: the preparation is oral liquid, and the oral liquid is prepared by the following steps: taking a polypeptide comprising SEQ ID NO: 2, adding a fructo-oligosaccharide solution with the concentration of 20% (w/v) or an L-arabinose solution with the concentration of 2% (w/v) to resuspend the bacteria to prepare 10 ml of bacteria ¹⁰ ～10 ¹² And (5) filling fresh bacterial liquid of the live cfu bacteria.

16. The medicament of claim 15, wherein: the number of viable bacteria in the oral liquid is 10 per gram ¹⁰ cfu。