CN110251661A

CN110251661A - It is a kind of for treating the pharmaceutical preparation of diabetes or weight-reducing

Info

Publication number: CN110251661A
Application number: CN201811520996.3A
Authority: CN
Inventors: 顾建文; 马婕; 马永平
Original assignee: Sichuan Litong Kechuang Biomedical Technology Co Ltd
Current assignee: Fuzhou Taijiang Xijiya Health Technology Co ltd
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2019-09-20
Anticipated expiration: 2038-12-12
Also published as: CN110251661B

Abstract

The invention discloses a kind of for treating the drug of diabetes or weight-reducing, it is using nucleotide sequence shown in amino acid sequence GLP-1 mutant as shown in SEQ ID NO:1, SEQ ID NO:2, the recombinant vector comprising nucleotide sequence shown in SEQ ID NO:2 and/or the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2 as effective component, in addition the preparation that pharmaceutically acceptable auxiliary material or complementary ingredient are prepared；The preparation is oral preparation.The present invention includes the drug of GLP-1 mutant, and blood sugar concentration or Weight-reducing and lipid-lowering can be effectively reduced, can be used for treating type-2 diabetes mellitus or obesity, potential applicability in clinical practice is good.

Description

It is a kind of for treating the pharmaceutical preparation of diabetes or weight-reducing

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of for treating the drug of diabetes or weight-reducing.

Background technique

With people's diet structure and living-pattern preservation, obesity has become worldwide epidemic disease.It is complete at present Ball wide-ultra weight and fat incidence are all in increased trend year by year.Especially China, overweight in recent years and fat hair Raw rate has been approached the level at even up to developed country's initial stage.For the crowd high for those BMI indexes, obesity can cause it The rising of his some diseases disease incidence, wherein being the most significantly diabetes, cardiovascular disease and cancer, overweight/obesity is serious Endanger the health of the mankind.

Meanwhile overweight/obesity be diabetes B (Type 2Diabetes Mellitus, T2DM) occur, development it is only Vertical risk factor, the two are the morbid states of associated interpromoting relation in five elements.Studies have shown that the every increase 1kg/m of constitutional index², the illness of T2DM Risk is increased by 12%；And in T2DM patient, overweight and obesity illness rate is more than 80%.Meanwhile overweight and fat shape The insulin-resistant states of T2DM patient can be further aggravated in state, and greatly increase the generation wind of the complication such as cardiovascular disease Danger.Specific income can be brought for T2DM patient by strengthening weight-reducing: being mitigated insulin-resistant states, repaired impaired beta Cell of islet Function, optimization glycemic control and the Related Risk Factors for improving cardiovascular disease etc..The fat vicious circle shape caused with T2DM State, make it is traditional with " hypoglycemic " be core treatment mode enter an awkward condition, this also opens more and more scholars Begin to pay close attention to the importance that " weight-reducing " is treated.

Although simple lifestyle modification can make the weight loss of obese patient, long-term efficacy is unsatisfactory, studies carefully its original On the one hand it is related to be difficult strict adherence weight-reducing life style with most of patient for cause；On the other hand also with most of conventional medicament It can inevitably put on weight in application process related.

But a variety of slimming medicines are while bringing good curative effect on obesity, because there is serious safety problems gradually It withdraws from the market.The slimming drug of clinical application at present mainly has the orlistat capsule of approval in 1999, the chlorine of approval in 2012 Ka Selin and phentermine and Topiramate capsule, Naltrexone Hydrochloride, Bupropion compound slow-release tablet and the benefit of approval in 2014 are drawn Shandong peptide injection.

Liraglutide (Liraglutide) is glucagon-like peptide 1 (glucagon-likepeptide-1, GLP-1) Similar peptide, for the novel drug based on secretin's effect.GLP-1 mainly passes through inhibition maincenter similar to peptide and ingests desire and suppression Gastrointestinal peristalsis processed increases satiety to reduce intake of the patient to energy, to play the curative effect of weight-reducing.Li Nalu peptide has Portugal The blood sugar reducing function of grape sugar concentration dependent, single therapy not will lead to hypoglycemia.

Liraglutide is in 2009 and 2010 respectively in USA and EU listing controlling for diabetes B (T2DM) It treats.2014 and 2015, FDA and European its supplement as diet control and physical training of drugs administration approved were used for The treatment of chronic fatty, for effect better than mainstream slimming drugs-orlistat in the market, fat-reducing effect is clear, is controlled for obesity First the daily primary GLP-1 treated is similar to peptide.Liraglutide is in 2011 in China's approval listing.Long term test shows benefit Draw Shandong peptide that can effectively mitigate patient's weight, adaptation population is the adult that body mass index (BMI) is greater than or equal to 30, or Be BMI be 27 or more, with the adult of at least one obesity complication such as T2DM, hypertension or high cholesterol.In people The existing slimming drugs of strong weight-reducing desire and unsatisfactory curative effect form big contradictory today, weight-reducing of the exploitation GLP-1 similar to peptides Medicine is also an only selection.Therefore, the drug of weight-reducing and hypoglycemic double benefit is had both with before very wide market Scape.

The GLP-1 including Liraglutide is both needed to drug administration by injection similar to galanin peptide drug at present, directly oral invalid, The disadvantages of inconvenient there are medication.But the treatment of diabetes usually requires long-term, continuous medication.The mode of drug administration by injection uses Inconvenient to carry, compliance is poor, it is also possible to which there are the risks such as irritation and allergic reaction, bring huge physiology to patient And mental anguish.And oral preparation is best suitable for the habit of people " clothes " medicine, preparation process is also mature and simple.Therefore from change GLP-1 starts with similar to the administration route of peptide, develops medication and easy to carry, the GLP-1 suitable for long-time service is similar to peptides medicine Object is significant.

Summary of the invention

The purpose of the present invention is to provide a kind of drugs comprising new GLP-1 mutant.

The present invention provides a kind of for treating the drug of diabetes or weight-reducing, it is with amino acid sequence such as SEQ ID GLP-1 mutant shown in NO:1, nucleotide sequence shown in SEQ ID NO:2, include nucleotide sequence shown in SEQ ID NO:2 Recombinant vector and/or recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2 be effective component, in addition can pharmaceutically connect The preparation that the auxiliary material or complementary ingredient received are prepared；The preparation is oral preparation.

Preferably, the oral preparation is tablet, capsule, pulvis, granule or oral solution.

Preferably, the pulvis is freeze dried powder.

Further, the freeze-dried powder is prepared as follows: being taken comprising nucleotide sequence shown in SEQ ID NO:2 The bacterial precipitation of recombinant bacterium adds the oligofructose solution that concentration is 20% (w/v) that bacterium is resuspended, and adding concentration is 15% (w/ V) glycerite, concentration are that the skimmed milk power solution of 10% (w/v), the lactose solution that concentration is 3% (w/v) and concentration are The aqueous trehalose of 3% (w/v), being configured to every gram is 10¹⁰~10¹²The mixing liquid of cfu viable bacteria is lyophilized after packing； In the mixing liquid, the volume of oligofructose solution, glycerite, skimmed milk power solution, lactose solution and aqueous trehalose Than for (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2:3:5:6:1；Preferably, it mixes for described every gram Closing the number of viable in liquid is 10¹⁰cfu。

Preferably, the pulvis is dry pulvis, the dry pulvis as follows: take comprising SEQ ID NO:2 The bacterial precipitation of the recombinant bacterium of shown nucleotide sequence adds concentration for the oligofructose solution resuspension bacterium of 20% (w/v), then plus Entering concentration is the skimmed milk power solution of 10% (w/v), the lactose solution that concentration is 3% (w/v) and sea that concentration is 3% (w/v) Algae sugar juice, being configured to every gram is 10¹⁰~10¹¹The temperature of the mixing liquid of cfu viable bacteria, drying, drying is no more than 40 DEG C；Institute State in mixing liquid, oligofructose solution, skimmed milk power solution, lactose solution and aqueous trehalose volume ratio be (1~10): (1~10): (1~10): (1~10), preferably 2:5:6:1；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

Preferably, the preparation be granule, the granule as follows: take comprising shown in SEQ ID NO:2 The bacterial precipitation of the recombinant bacterium of nucleotide sequence adds the oligofructose solution that concentration is 20% (w/v) that bacterium is resuspended, adds dense It is molten to spend the glycerite for being 15% (w/v), the lactose that concentration is the skimmed milk power solution of 10% (w/v), concentration is 3% (w/v) The aqueous trehalose that liquid and concentration are 3% (w/v), being configured to every gram is 10¹⁰~10¹²The mixing liquid of cfu viable bacteria, it is dry, it obtains Pulvis, is added the corrigent of 0.5~2 times of weight, the magnesium stearate of 0.5~2 times of weight, mixes, and granulation obtains granule；It is described In mixing liquid, oligofructose solution, glycerite, skimmed milk power solution, lactose solution and aqueous trehalose volume ratio be (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2:3:5:6:1；Preferably, every gram of mixed liquor Number of viable in body is 10¹⁰cfu。

Preferably, the preparation be tablet, the tablet as follows: take comprising nucleosides shown in SEQ ID NO:2 The bacterial precipitation of the recombinant bacterium of acid sequence adds the oligofructose solution that concentration is 20% (w/v) that bacterium is resuspended, and adding concentration is The glycerite of 15% (w/v), concentration are the skimmed milk power solution of 10% (w/v), concentration is 3% (w/v) lactose solution and Concentration is the aqueous trehalose of 3% (w/v), and being configured to every gram is 10¹⁰~10¹²The mixing liquid of cfu viable bacteria, it is dry, obtain powder Agent, is added the corrigent of 0.5~2 times of weight, the magnesium stearate of 0.5~2 times of weight, mixing, and tabletting obtains tablet.

Preferably, in the mixing liquid, oligofructose solution, glycerite, skimmed milk power solution, lactose solution and sea The volume ratio of algae sugar juice is (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2:3:5:6:1；It is excellent Selection of land, the number of viable in every gram of mixing liquid are 10¹⁰cfu。

Preferably, the preparation be capsule, the capsule as follows: take comprising shown in SEQ ID NO:2 The bacterial precipitation of the recombinant bacterium of nucleotide sequence adds the oligofructose solution that concentration is 20% (w/v) that bacterium is resuspended, adds dense It is molten to spend the glycerite for being 15% (w/v), the lactose that concentration is the skimmed milk power solution of 10% (w/v), concentration is 3% (w/v) The aqueous trehalose that liquid and concentration are 3% (w/v), being configured to every gram is 10¹⁰~10¹²The mixing liquid of cfu viable bacteria, it is dry, it obtains Pulvis is added the corrigent of 0.5~2 times of weight, the magnesium stearate of 0.5~2 times of weight, mixes, and granulation obtains granule, fills glue Capsule obtains capsule；In the mixing liquid, oligofructose solution, glycerite, skimmed milk power solution, lactose solution and seaweed The volume ratio of sugar juice is (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2:3:5:6:1；It is preferred that Ground, the number of viable in every gram of mixing liquid are 10¹⁰cfu。

Preferably, the preparation be oral solution, the oral solution as follows: take comprising core shown in SEQ IDNO:2 The bacterial precipitation of the recombinant bacterium of nucleotide sequence, adding oligofructose solution that concentration is 20% (w/v) or concentration is 2% (w/v) L-arabinose solution bacterium is resuspended, be configured to every milliliter not less than 10¹⁰~10¹²The fresh bacterium solution of cfu viable bacteria, it is filling, i.e., It can；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

Compared with natural GLP-1, GLP-1 mutant of the present invention reduces the degradation of DPP-IV, and half-life period is about 3h；And the recombinant probiotics containing GLP-1 mutant that the present invention constructs, it can be prepared into a plurality of types of solids and liquid system Oral administration is realized in agent, avoids the pain of patient's long term injections medication；Meanwhile after human body is taken, which can With field planting of surviving in human body intestinal canal, become functional in-vivo biological reactor, persistently generate and secrete GLP-1 mutant and is more Peptide, to play the effect of lasting blood sugar reducing function and treatment obesity, potential applicability in clinical practice is good.

Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.

The specific embodiment of form by the following examples remakes further specifically above content of the invention It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention The technology realized all belongs to the scope of the present invention.

Detailed description of the invention

The selection result of GLP-1GM mutant protein in Fig. 1 present invention.

GLP-1GM mutant SDS-PAGE and WB testing result Fig. 2 of the invention.

The hypoglycemic effect of the GLP-1GM mutant stomach-filling T2DM rat model of Bacillus coli expression in Fig. 3 present invention.

The hypoglycemic effect of the GLP-1GM mutant stomach-filling T2DM rat model of lactic acid bacteria bacterium expression in Fig. 4 present invention.

Fig. 5 mutant GM converts influence of the Escherichia coli Nissle1917 to mouse weight and weight gain value, and normal Group compares Δ p < 0.05, Δ Δ p < 0.01；The * p < 0.05 compared with model group, * * p < 0.01.

Fig. 6 mutant GM converts influence of the Escherichia coli Nissle1917 to mouse food ration, the Δ p < compared with normal group 0.05, Δ Δ p < 0.01；The * p < 0.05 compared with model group, * * p < 0.01.

Fig. 7 mutant GM convert Escherichia coli Nissle1917 to mouse fasting blood-glucose and blood sugar concentration change curve below Long-pending influence, Δ p < 0.05, Δ Δ p < 0.01 compared with normal group；The * p < 0.05 compared with model group, * * p < 0.01.

Fig. 8 mutant GM converts Escherichia coli Nissle1917 to mouse kidney peripheral adipose tissue weight and rouge body ratio It influences, Δ p < 0.05, Δ Δ p < 0.01 compared with normal group；The * p < 0.05 compared with model group, * * p < 0.01.

Fig. 9 mutant GM converts Escherichia coli Nissle1917 to mouse testis (ovary) peripheral adipose tissue weight and rouge The influence of body ratio, Δ p < 0.05, Δ Δ p < 0.01 compared with normal group；The * p < 0.05 compared with model group, * * p < 0.01.

Figure 10 mutant GM converts influence of the Escherichia coli Nissle1917 to mouse liver weight, the Δ compared with normal group P < 0.05, Δ Δ p < 0.01；The * p < 0.05 compared with model group, * * p < 0.01.

Specific embodiment

It is described further below with embodiment, but the present invention is not limited to these embodiments.

Experiment reagent used in the present invention and instrument are as follows:

PGEX-4T-1, pMG36e, pET32a, E.coli TOP10 and E.coli BL21 (DE3), Escherichia coli Nissle 1917, the bacterial strains such as Bacillus acidi lactici, Lactococcus lactis and plasmid are by Chengdu Li Lai Biotechnology Co., Ltd and Medical University Of Chongqing Biochemistry and Molecular Biology teaching and research room saves；Male rat is provided by Medical University Of Chongqing's Experimental Animal Center.T4 connection Enzyme, Taq normal enzyme, Sal I, BamH I, protein Marker, DNA Marker, Plasmid Mini kit I, Cycle-Pure Kit, plastic recovery kit, IPTG, erythromycin, ampicillin, urea assistant rhzomorph, BCA determination of protein concentration kit, PMSF etc. Reagent (being commercially available).

The screening of the mutant sequence of the present invention of embodiment 1 and Activity determination

From GLP-1 original series, 4 mutant polypeptide sequences of improvement and design, to pass through chemical synthesis (middle peptide is raw Change), obtain the polypeptide product that purity is up to 85%.Polypeptide sequence is as follows:

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

GM (SEQ ID NO:1):

HHEGTFTSDVSSYLEGQAAKKFIAWLVRGGKKKKKYGRKKRRQRRREF

C33:HACGTFTSDVSSYLEGQAAKEFIAWLCKGRG

K34:HAEGTFTSDVSSYLEGQAAKEFIAWLVKKKK

R34:HAEGTFTSDVSSYLEGQAAKEFIAWLVRRRR

GM is on the basis of GLP-1 original series, is histidine, 27 paddy ammonia by the 8th alanine mutation of GLP-1 Acid mutation is lysine, and 34 lysine mutations are arginine, and 36 arginine sport glycine, 37 glycine Lysine is sported, and a Duan Xulie is added in end.

C33 is on the basis of GLP-1 original series, is cysteine, 33 figured silk fabrics by the 9th glutamic acid mutation of GLP-1 Histidine mutations are cysteine.

K34 is on the basis of GLP-1 original series, is lysine, the 36th essence by the 35th glycine mutation of GLP-1 Histidine mutations are lysine, the 37th glycine mutation is lysine.

R34 is on the basis of GLP-1 original series, is arginine by the 34th lysine mutation of GLP-1, the 35th sweet Histidine mutations are arginine, and the 37th glycine mutation is arginine.

GLP-1 original series and the difference of mutant R34, K34 and C33 are as shown in table 1.

The sequence of table 1GLP-1 and mutant compares

Male rat is taken, every group 6, fasting 12h, after measuring blood glucose, 20mmol/kg glucose, rear abdominal cavity is injected intraperitoneally Aforementioned four 50 μ g/kg of mutant polypeptide is given in injection.Each time point measures blood glucose with three promise blood glucose meters (GA-3 type) after administration.

As a result as shown in Figure 1.

Easily by dipeptidyl peptidase IV (DPP-IV) rapid hydrolytic inactivation, half-life period is less than 5min, is unsuitable for natural GLP-1 Clinical application, therefore this zoopery is not carried out.

As it can be seen that GLP-1 mutant (GLP-1GM) hypoglycemic activity that number is GM is preferable, and long half time is up to 3 hours, and Other GLP-1 mutant hypoglycemic abilities and half-life period are poor.

The expression of recombinant e. coli of the mutant of the present invention of embodiment 2 and activity verifying

1, the Bacillus coli expression of GLP-1 mutant of the present invention

(1) a segment signal peptide sequence is added before the sequence of GLP-1 mutant GM, HIV cell-penetrating peptide sequence is added at 3 '-ends With 6 His-tag sequences, constructing being capable of secreting, expressing and with the GLP-1 mutant across cell membrane ability, nucleotide Sequence is as shown in SEQ ID NO:2.

SEQ ID NO:2:

atgaaaaagaacatcgcattcctcctggcatctatgtttgttttctctatcgctaccaacgcttacgc tggatcccaccacgagggcaccttcacctccgacgtgtcctcctacctggagggccaggccgccaagaagttcatc gcctggctggtgcgcggcggcaagaagaagaagaagtacggccgcaagaagcgccgccagcgccgccgcctcgagg acgacgacgacaagcaccatcaccatcaccattaa

(2) by nucleotide sequence shown in SEQ ID NO:3 be cloned into coli expression carrier pGEX BamHI and The site SalI obtains pGEX-GLP-1GM carrier, is sequenced in correct rear conversion E. coli BL21 (DE3), purpose piece Duan great little is about 12KD.Western blot identifies that expression of results is correct after nickeliferous magnetic beads for purifying, as a result as shown in Figure 2.It can See, successful expression of the present invention GLP-1 mutant protein.

(3) pGEX-GLP-1GM expression vector is transformed into Escherichia coli Nissle 1917, is identified through PCR and sequencing PGEX-GLP-1GM is converted successfully.Recombination bacillus coli group bacterium Nissle 1917 is taken, is inoculated into added with final concentration of 100 μ g/ On the LB culture medium of the ampicillin of ml, 37 DEG C, 250r/min shaking table culture to OD₆₀₀Value reaches 0.8~1.0, bacterium solution centrifugation After abandon supernatant, precipitating PBS tune OD₆₀₀It is worth 1.2.

3, the activity verifying of GLP-1 mutant

According to every Oral Administration in Rats stomach-filling 1ml recombination bacillus coli (OD₆₀₀=1.2) processing STZ induces successful T2DM big Mouse model, every group of 6 rats, each time point after stomach-filling, docking take blood, measure a blood using three promise blood glucose meters (GA-3 type) Sugar, to determine the hypoglycemic effect of oral GLP-1GM mutant.As a result as shown in Figure 3.

As it can be seen that 2 hours after the GLP-1GM mutant stomach-filling expressed in Escherichia coli Nissle 1917 have hypoglycemic effect (compared with the control group of non-stomach-filling, P < 0.01).Blood glucose slightly gos up after stomach-filling 8 hours.

The preparation of 3 mutant GM of embodiment conversion Escherichia coli Nissle1917 preparation

1, mutant GM converts the preparation of Escherichia coli Nissle1917 oral solution

Added with concentration be 200 μ g/ml Erythromycinresistant LB liquid medium steam sterilizing after, the PCR that learns from else's experience verifying and The Escherichia coli Nissle1917 (preparation of embodiment 2) of the gene stable conversion of inducing expression experimental identification is inoculated with, 37 DEG C, After 250r/min shaking table culture reaches 0.8~1.0 to OD600 value, centrifugation 5min harvests bacterium, with brine 2 times, from After the heart collects bacterial sediment, concentration is added to be resuspended carefully for the L-arabinose solution of 2% (w/v) or 20% oligofructose solution Bacterium, being configured to every milliliter is 10¹⁰The fresh bacterium solution of cfu viable bacteria saves after filling in 4 DEG C of refrigerator cold-storages.

2, mutant GM converts the preparation of Escherichia coli Nissle1917/ freeze dried powder

The bacterial precipitation for taking the above method to harvest adds the oligofructose that concentration is 20% (w/v) that bacterium is resuspended, adds dense Spend the glycerol for being 15% (w/v), concentration is that the skimmed milk power of 10% (w/v), the lactose that concentration is 3% (w/v) and concentration are 3% (w/v) trehalose, being configured to every gram is 10¹⁰The mixing liquid of cfu viable bacteria, in mixing liquid, the oligofructose of 20% (w/v) Solution, the glycerite of 15% (w/v), the skimmed milk power solution of 10% (w/v), 3% lactose solution, 3% trehalose are molten The volume ratio of liquid is 2:3:5:6:1, is placed in after packing on freeze drier and is lyophilized, and 4-8 DEG C of refrigerator saves after sealing.

3, mutant GM converts the preparation of the dry pulvis of Escherichia coli Nissle1917

The bacterial precipitation for taking the above method to harvest adds the oligofructose that concentration is 20% (w/v) that bacterium is resuspended, adds dense Degree is skimmed milk power, the lactose of 3% (w/v) and the trehalose of 3% (w/v) of 10% (w/v), is configured to every gram 10¹⁰Cfu viable bacteria Mixing liquid, in mixing liquid, the oligofructose solution of 20% (w/v), the skimmed milk power solution of 10% (w/v), 3% cream Sugar juice, 3% aqueous trehalose volume ratio be 2:5:6:1, be placed in after packing vacuum drying instrument on dry, maximum temperature is not More than 40 DEG C, 4-8 DEG C of refrigerator is saved after sealing.

4, mutant GM converts the preparation of Escherichia coli Nissle1917 granule

0.5~2 part of corrigent, flavoring is added in conversion Escherichia coli Nissle1917 freeze-dried powder obtained/drying pulvis Agent includes sweetener or fruity aromatic, uniformly mixes 20-30min, and 10-100 purpose is collected in investment dry granulating machine granulation Conversion Escherichia coli Nissle1917 granule is obtained after particle packing.

5, mutant GM converts the preparation of Escherichia coli Nissle1917 tablet

By Escherichia coli Nissle1917 freeze-dried powder obtained above and 0.5~2 part of corrigent, 0.5~2 part of magnesium stearate It is placed in 5~15min of mixing in powder mixer, the raw material of directly compressible is obtained after mixing, is placed in single-punching tablet press Tabletting, the Escherichia coli Nissle1917 tablet converted.

6, mutant GM converts the preparation of Escherichia coli Nissle1917 capsule

It is by Escherichia coli Nissle1917 particle obtained, the particle is filling in empty hard capsule by dimension, Gained capsule is placed in polishing machine to the Escherichia coli Nissle1917 capsule that extra dust of skimming is converted.

4 mutant GM of embodiment converts lactic acid bacteria

1, mutant GM converts lactic acid bacteria

(1) by GLP-1GM, i.e. nucleotide sequence shown in SEQ ID NO:2 is building up to lactic acid bacteria expression vectors pMG36e The site SalI and HindIII on plasmid, after electrotransformation Bacillus acidi lactici, the single colonie of picking recombinant Lactobacillus is extracted after culture Plasmid, PCR is examined and sequencing detection GLP-1GM gene is present in this receptor bacterium.

(2) above-mentioned recombinant lactic acid bacteria is inoculated on the MRS culture medium added with the erythromycin that concentration is 20 μ g/ml, 30 DEG C of stationary cultures are to OD₆₀₀When value reaches 0.8~1.0, centrifugation abandons supernatant, takes thallus, spare.

The preparation of 5 mutant GM of embodiment conversion lactobacillus preparation

1, mutant GM converts the preparation of lactic acid bacteria oral liquor

MRS fluid nutrient medium steam sterilizing added with the erythromycin that concentration is 20 μ g/ml, the PCR that learns from else's experience verifying and induction Lactic acid bacteria (preparation of the embodiment 5) inoculation of the gene stable conversion of experimental identification is expressed, 30 DEG C of stationary cultures to OD600 value reach When 0.8~1.0, centrifugation, abandon supernatant, take bacterial sediment add concentration be 2% (w/v) L-arabinose solution or 20% it is oligomeric Bacterium is resuspended in fructose soln, is configured to every milliliter not less than 10¹⁰The fresh bacterium solution of viable bacteria saves after packing in 4 DEG C of refrigerator cold-storages.

2, mutant GM converts the preparation of lactic acid bacteria freeze drying powder

With embodiment 3.

3, mutant GM converts the preparation of the dry pulvis of lactic acid bacteria

With embodiment 3.

4, mutant GM converts the preparation of Escherichia coli Nissle1917 granule

With embodiment 3.

5, mutant GM converts the preparation of Escherichia coli Nissle1917 tablet

With embodiment 3.

6, mutant GM converts the preparation of Escherichia coli Nissle1917 capsule

With embodiment 3.

Beneficial effects of the present invention are proved below by way of the mode of test example:

The recombinant lactic acid bacteria of the mutant of the present invention of experimental example 1 is expressed and activity verifying

1, the lactic acid bacteria expression of GLP-1 mutant of the present invention

2, the activity verifying of GLP-1 mutant

According to every rat oral gavage 1ml recombinant lactic acid bacteria OD₆₀₀=1.2, the T2DM animal model of processing STZ induction, every group 6 rats, each time point after stomach-filling, docking take blood, a blood glucose are measured using three promise blood glucose meters (GA-3 type), to determination The hypoglycemic effect of oral recombinant lactic acid bacteria-GLP-1GM mutant.

As a result as shown in Figure 4.In Fig. 4, the hypoglycemic effect after 26 hours is the hypoglycemic effect of 2h after mending hello recombinant lactic acid bacteria for 24 hours Fruit.

As it can be seen that the GLP-1GM mutant expressed in recombinant lactic acid bacteria has certain hypoglycemic effect, and in Escherichia coli As a result similar, the half-life period of GLP-1GM is still shorter, stomach-filling blood sugar concentration decline (P < 0.01) in 2 hours, blood glucose after 5-7 hours Slightly go up (P < 0.05), hypoglycemic effect weakens for 24 hours, if the recombinant lactic acid bacteria of secondary stomach-filling equivalent immediately, after 2h (i.e. 26h) obtain hypoglycemic effect (P < 0.01) again.

Therefore, the GLP-1GM mutant expressed in recombinant lactic acid bacteria equally has orally-taken blood sugar reducing effect.

2 mutant GM of experimental example converts the research of Escherichia coli Nissle1917 antiobesity action

1, experimental animal

SPF grades of kunming mices, weight 20-25g test Company of Animals Ltd. up to rich fruit purchased from Chengdu.Quality certification number: SCXK (river) 2015-030.Animal is quarantined and adaptability observation 3 days after receiving, and starts to test after qualified.Animal free water is taken the photograph Food, temperature (20 ± 2) DEG C, humidity (60 ± 5) %, 12h periodicity of illumination.

2, experimental animal is grouped

Experimental animal is randomly divided into: normal group, model group, recombination bacillus coli Nissle1917 (preparation of embodiment 5) are high Middle low dose group, every group of 10 animals.

3, experimental animal modeling and medication

Normal group feeding normal diet, remaining four groups of feeding high-sugar-fat-diet (feed formula are as follows: normal diet 67%, Sucrose 20%, lard 10%, cholesterol 2%, sodium taurocholate 1%).The daily ad lib of experimental animal and drinking-water.Recombinate large intestine bar The high, normal, basic dosage group difference stomach-filling viable count of bacterium Nissle1917 is 10¹⁰、10⁹、10⁸Each 0.3ml of CFU bacterium solution, model group stomach-filling Isometric sterile saline, once a day, respectively in 4th week and materials measurement in the 8th week.

4, Testing index

(1) weight: set time measurement experiment the weight of animals weekly, and calculate weight gain value.

(2) food ration: weekly the set time measure every group of experimental animal to appetite and surplus appetite, calculate food ration.

(3) the 8th week, it is empty that body weight determination fasting blood-glucose and oral glucose tolerance (OGTT) measurement: is weighed after animal fasting 12h After abdomen blood glucose, the glucose solution 3g/kg that the disposable stomach-filling concentration of every mouse is 50%, 30min after stomach-filling glucose, 60min, 120min, 180min tail point take blood to measure each time point blood glucose value using three promise blood glucose meters (GA-3 type), and calculate song Area (AUC) under line.

(4) when liver weight measures for body fat weight, rouge body: after experiment, putting to death animal, removes liver, kidney All adipose tissues and testis (ovary) peripheral adipose tissue are simultaneously weighed.It calculates separately around perirenal adipose tissue and testis (ovary) Adipose tissue and the ratio of weight, that is, rouge body ratio.Liver is removed simultaneously, weighs liver weight.

5, experimental result

(1) weight and weight gain value

As shown in figure 5, model group body weight and weight gain value more normally organize significant raising (p < 0.05).With model group It compares, bacterium solution senior middle school low dose group can significantly reduce the weight and weight gain value (p < 0.05) of experimental animal when surrounding, and eight The high middle dose group of bacterium solution can significantly reduce the weight and weight gain value (p < 0.05) of experimental animal when all.

(2) food ration

As shown in fig. 6, bacterium solution senior middle school low dose group can significantly reduce the food ration (p of experimental animal compared with model group < 0.05).Compared with normal group, there was no significant difference (p > 0.05) for the food ration of bacterium solution senior middle school low dose group.

(3) blood glucose and oral glucose tolerance

As shown in fig. 7, there was no significant difference (p > 0.05) for fasting blood-glucose between each group.After stomach-filling glucose solution, except normal Group is outer, and each group blood glucose significantly increases.Each group blood sugar concentration AUC is calculated, compared with model group, the high middle dose group of bacterium solution can be shown Write the AUC (p < 0.05) for reducing experimental animal carbohydrate tolerance test.

(4) body fat quality and rouge body ratio

As shown in figure 8, the kidney peripheral adipose tissue and rouge body of model group more normally organize significant raising (p < 0.05).Compared with model group, when 4th week, bacterium solution high dose group can significantly reduce the kidney peripheral adipose tissue of experimental animal And rouge body ratio (p < 0.05)；At the 8th week, the high middle dose group of bacterium solution can significantly reduce the kidney peripheral adipose group of experimental animal It knits and rouge body ratio (p < 0.05).

As shown in figure 9, testis (ovary) peripheral adipose tissue and rouge body of model group more normally organize significant raising (p < 0.05).Compared with model group, bacterium solution high dose group can significantly reduce testis (ovary) surrounding rouge of experimental animal when 4th week Fat tissue and rouge body ratio (p < 0.05)；The high middle dose group of bacterium solution can significantly reduce the testis (ovum of experimental animal at the 8th week Nest) peripheral adipose tissue and rouge body ratio (p < 0.05).

(5) liver weight

As shown in Figure 10, the liver organization weight ratio of model group more normally organizes significant raising (p < 0.05).With model group It compares, bacterium solution high dose group can significantly reduce the liver weight (p < 0.05) of experimental animal.

The above results show that mutant GM conversion Escherichia coli Nissle1917 can inhibit the appetite of experiment mice, reduce Energy absorption；The weight, body fat content, rouge body of experiment mice are significantly reduced than the weight with liver, is had fairly obvious Weight-reducing and lipid-lowering effect.The conversion Escherichia coli Nissle1917 can also significantly reduce blood glucose in oral glucose tolerance test simultaneously The area under the curve of variation has the trend of certain reduction blood glucose.

SEQUENCE LISTING

<110>Sichuan benefit leads to Kechuang biological medicine Science and Technology Ltd.

<120>a kind of for treating the pharmaceutical preparation of diabetes or weight-reducing

<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 segment

<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 kind of for treating the drug of diabetes or weight-reducing, it is characterised in that: it is with amino acid sequence such as SEQ ID NO:1 Nucleotide sequence shown in shown GLP-1 mutant, SEQ ID NO:2, the weight comprising nucleotide sequence shown in SEQ ID NO:2 Organizing carrier and/or the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2 is effective component, in addition pharmaceutically acceptable The preparation that auxiliary material or complementary ingredient are prepared；The preparation is oral preparation.

2. drug according to claim 1, it is characterised in that: the oral preparation is tablet, capsule, pulvis, particle Agent or oral solution.

3. drug according to claim 2, it is characterised in that: the pulvis is freeze dried powder.

4. drug according to claim 3, it is characterised in that: the freeze-dried powder is prepared as follows: being taken comprising SEQ The bacterial precipitation of the recombinant bacterium of nucleotide sequence shown in ID NO:2 adds the oligofructose solution that concentration is 20% (w/v) to be resuspended thin Bacterium, adds that concentration is the glycerite of 15% (w/v), concentration is the skimmed milk power solution of 10% (w/v), concentration is 3% (w/ V) aqueous trehalose that lactose solution and concentration is 3% (w/v), being configured to every gram is 10¹⁰~10¹²The mixed liquor of cfu viable bacteria Body is lyophilized after packing；In the mixing liquid, oligofructose solution, glycerite, skimmed milk power solution, lactose solution Volume ratio with aqueous trehalose is (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2:3:5:6: 1；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

5. drug according to claim 2, it is characterised in that: the pulvis is dry pulvis, the dry pulvis according to Following method: taking the bacterial precipitation of the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2, and adding concentration is 20% (w/v) Oligofructose solution be resuspended bacterium, adding concentration is the skimmed milk power solution of 10% (w/v), the cream that concentration is 3% (w/v) The aqueous trehalose that sugar juice and concentration are 3% (w/v), being configured to every gram is 10¹⁰~10¹²The mixing liquid of cfu viable bacteria is dried Dry, the temperature of drying is no more than 40 DEG C；In the mixing liquid, oligofructose solution, skimmed milk power solution, lactose solution and sea The volume ratio of algae sugar juice is (1~10): (1~10): (1~10): (1~10), preferably 2:5:6:1；Preferably, described every Number of viable in gram mixing liquid is 10¹⁰cfu。

6. drug according to claim 2, it is characterised in that: the preparation is granule, and the granule is according to as follows Method: taking the bacterial precipitation of the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2, and adding concentration is the low of 20% (w/v) Bacterium is resuspended in Fructooligosaccharides solution, and adding concentration is the glycerite of 15% (w/v), the skimmed milk power that concentration is 10% (w/v) Solution, the lactose solution that concentration is 3% (w/v) and aqueous trehalose that concentration is 3% (w/v), being configured to every gram is 10¹⁰~ 10¹²The mixing liquid of cfu viable bacteria, it is dry, pulvis is obtained, the corrigent of 0.5~2 times of weight, the tristearin of 0.5~2 times of weight is added Sour magnesium mixes, and granulation obtains granule；In the mixing liquid, oligofructose solution, glycerite, skimmed milk power solution, cream The volume ratio of sugar juice and aqueous trehalose is (1~10): (1~10): (1~10): (1~10): (1~10), preferably 2: 3:5:6:1；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

7. drug according to claim 2, it is characterised in that: the preparation be tablet, the tablet as follows: The bacterial precipitation for taking the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2, adding concentration is the oligofructose of 20% (w/v) Bacterium is resuspended in solution, adds concentration is the glycerite of 15% (w/v), concentration is 10% (w/v) skimmed milk power solution, dense The aqueous trehalose that the lactose solution and concentration that degree is 3% (w/v) are 3% (w/v), being configured to every gram is 10¹⁰~10¹²Cfu is living The mixing liquid of bacterium, it is dry, pulvis is obtained, the corrigent of 0.5~2 times of weight, the magnesium stearate of 0.5~2 times of weight is added, mixes It closes, tabletting obtains tablet.

8. obtaining drug according to claim 7, it is characterised in that: in the mixing liquid, oligofructose solution, glycerol are molten Liquid, skimmed milk power solution, lactose solution and aqueous trehalose volume ratio be (1~10): (1~10): (1~10): (1~ 10): (1~10), preferably 2:3:5:6:1；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

9. drug according to claim 2, it is characterised in that: the preparation is capsule, and the capsule is according to as follows Method: taking the bacterial precipitation of the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2, and adding concentration is the low of 20% (w/v) Bacterium is resuspended in Fructooligosaccharides solution, and adding concentration is the glycerite of 15% (w/v), the skimmed milk power that concentration is 10% (w/v) Solution, the lactose solution that concentration is 3% (w/v) and aqueous trehalose that concentration is 3% (w/v), being configured to every gram is 10¹⁰~ 10¹²The mixing liquid of cfu viable bacteria, it is dry, pulvis is obtained, the corrigent of 0.5~2 times of weight, the tristearin of 0.5~2 times of weight is added Sour magnesium mixes, and granulation obtains granule, encapsulated, obtains capsule；In the mixing liquid, oligofructose solution, glycerite, The volume ratio of skimmed milk power solution, lactose solution and aqueous trehalose is (1~10): (1~10): (1~10): (1~10): (1 ~10), preferably 2:3:5:6:1；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。

10. drug according to claim 2, it is characterised in that: the preparation is oral solution, and the oral solution is according to as follows Method: taking the bacterial precipitation of the recombinant bacterium comprising nucleotide sequence shown in SEQ ID NO:2, and adding concentration is the low of 20% (w/v) Bacterium is resuspended in the L-arabinose solution that Fructooligosaccharides solution or concentration are 2% (w/v), is configured to every milliliter not less than 10¹⁰~ 10¹²The fresh bacterium solution of cfu viable bacteria, it is filling；Preferably, the number of viable in every gram of mixing liquid is 10¹⁰cfu。