Probiotics for producing glucagon-like peptide-1 analogs and application thereof

Abstract

The present invention relates to the field of biomedical technology, and in particular to a probiotic that produces a glucagon-like peptide-1 analog and its application in the treatment of type 2 diabetes (T2DM) and obesity. E. coli Nissle1917 The engineered bacteria can secrete a glucagon-like peptide-1 (GLP-1) analogue that can penetrate the intestinal barrier, wherein the engineered bacteria knock out the membrane protein LPP gene, and the GLP-1 analogue that can penetrate the intestinal barrier is shown in SEQ ID NO.2. The present invention successfully constructs a glucagon-like peptide-1 (GLP-1) analogue that can secrete a glucagon-like peptide-1 (GLP-1) analogue that can penetrate the intestinal barrier. Nissle1917 ΔLPP (EcN‑P) can be applied to the development of fermentation strains, the preparation of probiotic tablets with weight loss function, and the production of drugs with blood sugar control function.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a probiotic for producing a glucagon-like peptide-1 analog and an application thereof, which is applied to the treatment of type 2 diabetes mellitus (T2DM) and obesity. Background Art

[0002] Probiotics are active microorganisms that are beneficial to the host. They can regulate the balance of intestinal flora, thereby enhancing the integrity of the intestinal epithelium, protecting the intestinal barrier, regulating the mucosal immune system, and inhibiting the growth of pathogenic bacteria. E. coli Nissle 1917 (EcN) was originally isolated by military surgeon Alfred Nissle from the feces of a soldier who did not suffer from diarrhea during a severe outbreak of shigellosis. In recent years, the intestinal probiotic EcN has been prepared into drugs for the treatment of diarrhea and ulcerative colitis.

[0003] Diabetes is a metabolic disease characterized by high blood sugar levels. Type 2 diabetes mellitus (T2DM) is caused by a progressive decrease in insulin secretion from pancreatic β cells in the context of insulin resistance, accounting for 90% to 95% of the diabetic population.

[0004] Glucagon-like peptide-1 (GLP-1) produced by intestinal goblet cells can reduce insulin resistance, enhance its sensitivity to insulin signals, alleviate T2DM, treat obesity and other effects. Glucagon-like peptide-1 (GLP-1) analogs, as a new type of T2DM treatment drug, are highly favored due to their significant efficacy and high safety in clinical application. However, although GLP-1 is derived from the human body, has significant efficacy and few side effects. However, there are sites in the amino acid sequence of GLP-1 that can be recognized by dipeptidyl peptidase IV (DPP-IV), which is easily degraded by DPP-IV in the body, with a half-life of only a few minutes, which greatly limits its physiological efficacy. In addition, GLP-1 can only be administered by injection, which makes many patients reluctant to take it.

[0005] Using probiotics as living drugs in biological factories can not only regulate the balance of intestinal flora, but also produce peptides and protein drugs through expression vectors to achieve disease intervention and treatment. However, the production of peptides and protein drugs by E. coli has problems such as low secretion capacity, product accumulation in cells, easy formation of inclusion bodies, and difficulty for drugs to penetrate the intestinal barrier. Summary of the invention

[0006] In view of the above-mentioned defects of the prior art, the present invention provides a glucagon-like peptide-1 (GLP-1) analogue that can colonize in the intestine and secrete a glucagon-like peptide-1 (GLP-1) that can penetrate the intestinal barrier. E.coli Nissle1917The engineered bacteria (EcN-P) has a knockout gene for the membrane protein LPP, and the GLP-1 analogue capable of penetrating the intestinal barrier is shown in SEQ ID NO.2.

[0007] Preferably, the intestinal barrier-penetrating GLP-1 analog comprises a transmembrane domain and a glucagon-like peptide functional domain.

[0008] Preferably, the transmembrane domain and the glucagon-like peptide functional domain are coupled by chemical synthesis.

[0009] Preferably, the knockout membrane protein LPP gene of the engineered bacteria is knocked out by lambda homologous recombination technology, and the nucleotide sequence after knockout is shown in SEQ ID NO.1.

[0010] Preferably, the LPP-F used for knocking out the LPP gene is shown as SEQ ID NO.4, and the Lpp-R is shown as SEQ ID NO.5.

[0011] Preferably, the engineered bacteria secrete the GLP-1 analog by introducing a high extracellular expression plasmid vector, and the plasmid vector also contains a signal peptide pleB and a strong promoter PJ23119.

[0012] In a preferred embodiment of the present invention, the present invention provides a pharmaceutical composition, the pharmaceutical composition comprising the present invention E.coli Nissle1917 Engineered bacteria (EcN-P).

[0013] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0014] In another preferred embodiment of the present invention, the present invention provides the E.coli Nissle1917 Application of engineered bacteria (EcN-P) and its pharmaceutical composition in the preparation of weight loss products.

[0015] In another preferred embodiment of the present invention, the present invention provides the E.coli Nissle1917 Application of engineered bacteria (EcN-P) and pharmaceutical compositions thereof in the preparation of drugs for treating type 2 diabetes and obesity.

[0016] The present invention successfully constructed a glucagon-like peptide-1 (GLP-1) analogue that can secrete and penetrate the intestinal barrier. Nissle1917 ΔLPP (EcN-P) can be applied to the development of fermentation strains, the preparation of probiotic tablets with weight loss function, and the production of drugs with blood sugar control function. It provides a basis for probiotic products with functions such as weight loss and blood sugar control, and has potential clinical applicability and research value in the treatment of type 2 diabetes (T2DM) and obesity.

[0017] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Highly exocytotic E.coli Nissle1917 Schematic diagram of the mechanism and construction process of engineered bacteria.

[0019] Figure 2 Show Nissle1917 ΔLPP Identification of the strain and the results of Sanger sequencing identification of the modified region.

[0020] Figure 3 This is a diagram of the structure of a high extracellular expression plasmid with a signal peptide pleB and a high expression promoter PJ23119.

[0021] Figure 4 yes Nissle1917 ΔLPP Growth and protein secretion ability test results.

[0022] Figure 5 This is the result diagram of the expression detection and purification of GLP-1 in engineered bacteria.

[0023] Figure 6 It is a result chart of carcass weight, lean body mass and fat weight of each part.

[0024] Figure 7 This is the pathological result of HE staining of important organs such as heart, liver, spleen, lung, and kidney. DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0026] Experimental Materials

[0027] Table 1 Primers used in the examples

[0028]

[0029] Embodiment 1: Nissle1917 ΔLPP Construction of strains

[0030] 1) Knockout fragment primer design

[0031] In Escherichia coli Nissle 191750bp of base sequences were selected from the upstream and downstream positions of the Braun lipoprotein gene in the genome of the strain 06: K5: H1 (serotype 06: K5: H1, NCBI sequence number: NZCP022686.1) as homologous arms for homologous recombination, and upstream and downstream amplification primers of the kanamycin resistance gene of the pKD4 vector were added to design LPP knockout primers LPP-F and LPP-R.

[0032] 2) Knockout fragment amplification

[0033] Using pKD4 as a template, LPP-F and LPP-R as primers, a high-fidelity enzyme was used to amplify the kanamycin resistance fragment with homology arms. The amplification system is shown in the figure below. After agarose gel electrophoresis and gel recovery, the knockout fragment LPP-KO was obtained, and the fragment sequence is shown in SEQ ID NO.3;

[0034] Components volume 2X Pfu PCR Mix 25μL LPP-F 2μL LPP-R 2μL pKD4 plasmid 1μL <![CDATA[ddH2O]]> 20μL Total 50μL

[0035] 3) Prepare EcN-pKD46 electroporation competent cells;

[0036] Take out the EcN-pKD46 strain stored in a -80°C refrigerator and activate and culture for 16-20 hours. Inoculate the fresh colony into 5 ml SOB medium and culture at 37°C with shaking overnight.

[0037] Transfer 250uL of the overnight culture into 50ml SOB medium and culture at 37℃ with vigorous shaking (300rpm) until OD600 is about 0.72-0.76. Place the bacterial culture in an ice-water mixture to quench.

[0038] Centrifuge at 4°C, 2000g for 10 min. Collect the cells and discard the supernatant. Gently resuspend the cells with pre-cooled sterile water, centrifuge at 4°C, 2200g for 10 min, discard the supernatant. Repeat the wash twice with 10% glycerol, centrifuge at 2400g for 10 min, discard the supernatant, and resuspend the cells with 10% glycerol.

[0039] 4) Knockout of target gene by electroporation:

[0040] The knockout fragment LPP-KO was electroporated into the above electroporation competent medium using an electroporator at a voltage of 2500 V, a resistance of 200 Ω, and a capacitance of 25 uF. After adding 0.2% arabinose for recovery for 6 hours, the plate was spread on a kanamycin-resistant plate and cultured at 37°C for 20 hours.

[0041] 5) Identification of highly exocytotic engineered bacteria

[0042] Select the 20bp bases 132bp upstream and 112bp downstream of the LPP homologous exchange site as identification primers LPP-tF and LPP-tR; pick the colonies on the kanamycin resistance plate, use LPP-tF and LPP-tR as primers, perform colony PCR to identify the insertion of the kanamycin resistance fragment, and the length of the PCR fragment of the successfully inserted colony is 1840bp; the sequencing identification is correct, which is Nissle1917 ΔLPP (EcN-P) engineered strain.

[0043] 6) Transformation and screening of GLP1 expression vector

[0044] Inoculate the correctly identified colony into 3mL LB culture medium and culture overnight. Inoculate into 50mL LB culture medium at 1:100 and shake vigorously at 37℃ until the OD600 of the culture medium reaches 0.375-0.4. Place the culture on ice for 10min, centrifuge at 4℃, 4000g for 15min, discard the supernatant, suspend the bacteria in 10-20mL pre-cooled 0.1mol / L CaCl2 solution, and place on ice for 20min. Centrifuge at 4℃, 4000g for 10min, discard the supernatant, then gently suspend the bacteria in 2ml 0.1mol / L CaCl2 solution, slowly drip sterilized glycerol to a final concentration of 15%, and gently mix.

[0045] Take 100µl of the shaken competent cell suspension, add 2µl (20ng) of PJ119 plasmid, mix gently, place on ice for 30min, heat shock in a 42℃ water bath for 90s, and then quickly cool on ice for 5min.

[0046] Add 900μl LB liquid medium to a total volume of about 1ml, mix well and culture at 37℃ with shaking for 90min to allow the recipient bacteria to recover to normal growth state and produce resistance. Take 30μL of bacterial solution and spread it evenly on the pre-made LB agar plate (Amp + , Kanr + ) and inverted for absorption at 37°C and cultured overnight. A single clone was selected and sequenced to identify the correct EcN-P; PJ119 engineering strain.

[0047] Experimental results: Figure 2 As shown in A, the identification fragment of EcN fimA , foc , Plasmid1 , Plasmid2 , p- Plasmid2 The sizes were all in the correct position, and the LPP sizes of EcN; PJ119 and EcN-P; PJ119 strains were in line with expectations. The LPP and ΔLPP fragments were recovered and Sanger sequenced as shown in Figure 2 The sequence shown in B was confirmed to be successfully knocked out.

[0048] Example 2: Production capacity detection of engineered strains and GLP-1 purification and identification

[0049] EcN, EcN; PJ119 and EcN-P; PJ119 strains were inoculated into LB medium supplemented with corresponding antibiotics and cultured overnight at 37°C and 250rpm. Inoculated into 100mL LB medium supplemented with corresponding antibiotics at a ratio of 1:100 and cultured at 37°C and 250rpm. The OD600 value of the culture medium was measured every 2 hours.

[0050] 1 mL of culture medium was collected every 2 hours, centrifuged at 4000 rpm and 4°C, and the bacteria and culture supernatant were collected separately, and the bacteria were resuspended in 100 μL PBS. The protein content of the culture supernatant and bacteria at different times was determined by the Barfod method. The bacteria were broken by ultrasound, and the culture supernatant was concentrated 20 times through a 10kD ultrafiltration tube. The His-Tag antibody was used to perform Western-Blot detection on the bacteria and supernatant to determine the expression of GLP-1.

[0051] The EcN-P; PJ119 strain was inoculated into LB medium supplemented with the corresponding antibiotics and cultured overnight at 37°C, 250rpm. A 1% inoculum was transferred into 100mL LB medium supplemented with the corresponding antibiotics and cultured at 37°C, 250rpm until the OD600 value of the medium reached 2.0.

[0052] Centrifuge at 4000g for 20 min at 4°C, collect the culture supernatant, concentrate 100 mL of the culture supernatant to 10 mL using a 10kD ultrafiltration tube, and then purify and separate the His-tagged GLP-1 using a Ni-NTA gravity column affinity chromatography column. The specific operation is as follows:

[0053] 1) Rinse the column

[0054] First, open the upper and lower covers of the gravity column to allow the ethanol to flow out naturally, and no residual ethanol should be visible on the surface.

[0055] Add 10 mL of Tris-HCl buffer and allow the buffer to flow out naturally and completely; add 10 mL of Tris-HCl buffer again and allow the buffer to flow out naturally and completely.

[0056] 2) Protein loading

[0057] Slowly add the supernatant after ultrafiltration and concentration to the gravity column to allow the protein sample to flow out naturally. Proteins containing His tags bind to the Ni-NTA medium, while proteins without His tags cannot bind to the affinity medium and therefore flow out directly. Add the effluent to the gravity column again to allow the tagged proteins to fully bind to Ni-NTA.

[0058] 3) Washing of impurity proteins

[0059] The proteins that cannot bind are further washed to make the obtained proteins purer.

[0060] Add 10 mL of 3 mM imidazole buffer and allow it to flow out naturally until it is completely out; add 10 mL of 3 mM imidazole buffer again and allow it to flow out naturally until it is completely out;

[0061] 4) Protein elution

[0062] The protein was eluted using a buffer containing a high concentration of imidazole. 5 mL of a buffer containing 40 mM imidazole was added and the effluent was collected.

[0063] 5) Protein ultrafiltration to remove imidazole

[0064] Use ultrafiltration tube to perform protein ultrafiltration to remove high concentration of imidazole: add the elution effluent to the ultrafiltration tube, centrifuge at 4000g for 60 minutes at 4°C to concentrate the protein to 0.5mL;

[0065] Discard the filtrate, add 10 mL of PBS buffer on top, and perform ultrafiltration again. Repeat 3-4 times to reduce the imidazole concentration by 1000 times.

[0066] 6) Yield and purity identification

[0067] The protein after removal of imidazole salt was mixed with loading buffer and heated at 95°C for 5 min to fully denature the protein. The lysate, flow-through, washing solution and eluate were detected by Western-blot using His-Tag antibody to determine the protein in the culture medium supernatant.

[0068] Experimental results: Figure 4 As shown in A, the growth rate of EcN-P; PJ119 is significantly lower than that of wild-type EcN and EcN; PJ119 strains. At the same time, the total protein content shows that when the bacterial density is the same, the protein content of EcN-P and EcN-P; PJ119 is significantly higher than that of wild-type EcN. Figure 4 B. There was no significant difference in the expression of extracellular proteins between EcN and EcN-P, both of which were much lower than EcN-P; PJ119, see Figure 4 C. As Figure 4As shown in D, the same OD600 samples were selected for Western-Blot detection in the three groups. No GLP-1 was detected in the culture supernatant and bacteria of wild-type EcN. The GLP-1 content in the culture supernatant and bacteria of EcN; PJ119 and EcN-P; PJ199 increased with time. Figure 4 As shown in C, GLP-1 was detected in the cell lysate, flow-through, washing solution, and elution solution. The results showed that high expression of GLP-1 was present in the supernatant. (It should be noted that Figure 4 The “M” in the figure stands for “Marker” in biological experiment data).

[0069] Example 3: Therapeutic effect of engineered Escherichia coli EcN-P on high-fat diet-induced obese mice

[0070] Preparation of strains: EcN, EcN; PJ119 and EcN-P; PJ119 were inoculated into LB medium containing the corresponding antibiotics, cultured at 37°C, 220 rpm until OD600 = 1.0, centrifuged at 5000g for 5 min, collected the cells, resuspended in sterile saline, and adjusted the cell count to 1×10 9 CFU / mL.

[0071] Eighteen 12-week-old C57 / BL6 male mice were selected and fed with 60% fat energy-supplying food (XHF60 from Synergy Biotech) to establish an obese mouse model. The mice were randomly divided into three groups: a. control group, n=6, who were given 100 μL of normal saline by gavage every week; b. wild-type mice Nissle 1917 Treatment group, n = 6, was given 100 μL (10 7 CFU) EcN; PJ119; c. Engineering Nissle 1917 Treatment group, n = 6, was given 100 μL (10 7 CFU) EcN-P; PJ119;

[0072] During the experiment, the weight changes of mice were measured and recorded every week, and blood sugar was tested after each administration. After 5 weeks of treatment, the mice were killed. Their organs and adipose tissues were removed, and the organs were stained with HE, and the fat weight of each part was weighed to evaluate the effect and safety of the treatment.

[0073] Experimental results: Figure 5 As shown in A, both EcN and EcN-P showed a certain blood sugar control effect during the 5-week treatment, and EcN-P was better than EcN. Figure 5 As shown in B, the weight of the Control group continued to increase, and both the EcN group and the EcN-P group had an anti-obesity effect. Weight results after 5 weeks of treatment Figure 5As shown in Figure C, both EcN and EcN-P showed a weight control effect, with EcN and EcN-P reducing body weight by 5% and 11%, respectively. After the animals were killed, subcutaneous fat and abdominal fat were separated and weighed accurately. The results are shown in Figure 3. Figure 6 Both EcN and EcN-P were shown to reduce fat accumulation. For subcutaneous fat, EcN and EcN-P reduced body weight by 21% and 33%, respectively. For abdominal fat, EcN and EcN-P reduced body weight by 29% and 21%, respectively. Total fat weight was reduced by 24% and 28%, respectively.

[0074] H&E staining of important organs Figure 7 As shown, no obvious tissue structural changes were observed. The results showed that both EcN and EcN-P had good safety when taken orally.

[0075] The preferred specific embodiments of the present invention are described in detail above. It should be understood that ordinary technicians in the field can make many modifications and changes based on the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by technicians in the technical field based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A glucagon-like peptide-1 (GLP-1) analogue that can colonize in the intestine and secrete a glucagon-like peptide-1 (GLP-1) that can penetrate the intestinal barrier E.coli Nissle1917 An engineered bacterium, wherein the membrane protein LPP gene is knocked out of the engineered bacterium, and the nucleotide sequence of the GLP-1 analogue capable of penetrating the intestinal barrier is shown in SEQ ID NO.2; The knockout membrane protein LPP gene of the engineered bacteria is knocked out by lambda homologous recombination technology, and the nucleotide sequence after the knockout is shown in SEQ ID NO.1; The nucleotide sequence of LPP-F used to knock out the membrane protein LPP gene is shown in SEQ ID NO.4, and the nucleotide sequence of LPP-R is shown in SEQ ID NO.

5.

2. according to claim 1 E.coli Nissle1917 The engineered bacteria is characterized in that The engineered bacteria secrete the GLP-1 analog by introducing a high extracellular expression plasmid vector, wherein the plasmid vector also contains a signal peptide pleB and a strong promoter PJ23119.

3. A pharmaceutical composition comprising the compound described in any one of claims 1 to 2 E.coli Nissle1917 Engineered bacteria.

4. The pharmaceutical composition according to claim 3, further comprising a pharmaceutically acceptable carrier or excipient.

5. The method according to any one of claims 1 to 2 E.coli Nissle1917 Application of engineered bacteria in the preparation of weight loss products.

6. The method according to any one of claims 1 to 2 E.coli Nissle1917 Use of the engineered bacteria and / or the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of drugs for treating type 2 diabetes and obesity.

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