Lactococcus lactis expressing engineered exendin-4 and construction method and application thereof

Abstract

The application discloses a recombinant Lactococcus lactis engineering bacterium expressing reformed Exendin-4 and a construction method and application thereof. The engineering bacterium is obtained by introducing a recombinant food-grade plasmid pNZ8149-UEm-His into Lactococcus lactis NZ3900, and the genotype is NZ3900 / pNZ8149-UEm-His. The reformed Exendin-4 is obtained by performing three site-directed mutations on natural Exendin-4, and the three site-directed mutations include: replacing asparagine at the 28th position with glutamine; eliminating a trypsin recognition site of Lys12-Arg13; and adding a cysteine residue at the C terminal. The engineering bacterium realizes high-efficiency secretory expression of the target protein based on a NICE induction system. The production system provided by the application has the advantages of food safety level, no anti-screening and simple process, and is suitable for development of an intestinal delivery system.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a modified Lactococcus lactis expressing Exendin-4, its construction method, and its application. Background Technology

[0002] Glucagon-like peptide-1 (GLP-1) analogs lower blood glucose through a glucose-dependent mechanism, while also suppressing appetite and slowing gastrointestinal motility, demonstrating good efficacy and safety in the treatment of type 2 diabetes and obesity. Currently, long-acting GLP-1 analogs used clinically (such as liraglutide and smegglutide) are all injectable formulations, which limit patient compliance, necessitating the development of oral alternatives.

[0003] Exendin-4 is a natural GLP-1 receptor agonist derived from the saliva of the Gila monster. Its resistance to degradation by dipeptidyl peptidase-4 (DPP-4) results in a significantly longer plasma half-life (approximately 2.4 hours) than endogenous GLP-1 (<2 minutes), and it has been successfully marketed for injectable therapy. However, Exendin-4 still has two inherent limitations that restrict its oral application: (1) the asparagine at position 28 (Asn28) is prone to deamidation, leading to chemical inactivation; and (2) the lysine-arginine combination at positions 12-13 (Lys12-Arg13) constitutes a trypsin-specific cleavage site, causing it to degrade rapidly in the intestine, resulting in extremely low oral bioavailability.

[0004] Currently, the production of Exendin-4 mainly relies on chemical synthesis or E. coli expression. The former is costly and difficult to scale up; the latter, although with high yield, is prone to inclusion body formation, and the active structure containing two pairs of disulfide bonds has low refolding efficiency, both of which are difficult to meet the demand for low-cost, high-purity raw materials for oral formulations.

[0005] Lactococcus lactis, a generally recognized safe (GRAS) food-grade probiotic, not only possesses excellent intestinal colonization potential but can also serve as a live bacterial delivery carrier for orally administered peptides. Engineered bacteria, after oral administration, can colonize the intestine and secrete active proteins in situ, thereby protecting them from gastric acid degradation and achieving local release. The Nisin-Controlled Gene Expression (NICE) system, induced by nisin A, enables the controlled expression of exogenous proteins under food-grade inducer (Nisin) conditions. However, existing Lactococcus lactis expression systems still face challenges in the production of Exendin-4 analogs, such as low expression levels and the susceptibility of the product to degradation by proteases (e.g., trypsin) in the intestinal environment.

[0006] Therefore, there is an urgent need for a strategy that combines rational protein engineering with a food-grade safe expression platform to develop a highly stable, degradation-resistant, and orally deliverable modified Exendin-4 production system to promote the transformation of GLP-1 therapy to non-injectable administration. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical bottlenecks of existing Exendin-4 in oral applications, such as chemical instability, easy degradation in the intestine, and insufficient safety of the production system. By performing three site-directed mutagenesis on Exendin-4, a modified Exendin-4 production platform with high stability, resistance to enzymatic degradation, and suitable for oral delivery is provided.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution.

[0009] In a first aspect, the present invention provides a recombinant Lactococcus lactis engineered bacterium expressing modified Exendin-4. The engineered bacterium is constructed by introducing the recombinant plasmid pNZ8149-UEm-His into Lactococcus lactis NZ3900, and its genotype is NZ3900 / pNZ8149-UEm-His.

[0010] The modified Exendin-4 was obtained by performing three site-directed mutations on natural Exendin-4 and contains the following three mutational characteristics: (1) Mutate asparagine (Asn), which is prone to deamidation reaction at position 28, to glutamine (Gln) to significantly improve its chemical stability; (2) By eliminating the trypsin-specific recognition site composed of lysine-arginine (Lys12-Arg13) at positions 12-13 through point mutation, its resistance to degradation in the intestinal environment is enhanced. (3) Add a cysteine ​​(Cys) residue at the C-terminus to promote intestinal mucosal absorption.

[0011] Preferably, the nucleotide sequence of the gene encoding the modified Exendin-4 is shown in SEQ ID No:1.

[0012] Furthermore, the recombinant plasmid pNZ8149-UEm-His is constructed based on the food-grade expression plasmid pNZ8149, which contains the PnisA promoter, the lacF selection marker gene, and the replication initiation proteins RepA and RepC.

[0013] A second aspect of the present invention provides a method for constructing the above-mentioned recombinant Lactococcus lactis engineered strain. The method includes the following steps: (1) Synthesize the gene fragment encoding the modified Exendin-4; (2) The gene fragment was cloned into the food-grade expression plasmid pNZ8149 to obtain the recombinant plasmid pNZ8149-UEm-His; (3) The recombinant plasmid was transformed into Lactococcus lactis NZ3900 competent cells to obtain the engineered bacteria.

[0014] Preferably, in step (2), the Kinmen cloning method is used to achieve efficient and seamless connection between gene fragments and vectors.

[0015] Preferably, in step (3), the recombinant plasmid is introduced into competent cells by electroporation, and positive clones are screened on lactose medium without antibiotics by utilizing the functional complementarity of the lacF gene.

[0016] A third aspect of the present invention provides a method for producing modified Exendin-4. The method comprises: fermenting and culturing the recombinant Lactococcus lactis engineered strain according to any one of claims 1 to 3 to obtain a culture containing the recombinant Lactococcus lactis engineered strain, and obtaining modified Exendin-4 from the culture.

[0017] Preferably, the fermentation is carried out in LLB-whey medium; when the cell OD... 600 When the concentration reaches 0.3-0.4, nisin A at a final concentration of 5-15 ng / mL is added for induction to initiate efficient secretory expression of the exogenous protein.

[0018] A fourth aspect of the present invention provides the potential application of the recombinant lactococcus engineered strain in the biomedical field.

[0019] The engineered bacteria can secrete and express modified Exendin-4, which retains GLP-1 receptor agonist activity. Based on the good intestinal tolerance and food safety characteristics of *Lactococcus lactis*, this engineered bacteria holds promise as an intestinal-targeted delivery vector, releasing modified Exendin-4 in the intestinal environment after oral administration, thus providing a novel strategy for the intervention of type 2 diabetes and related metabolic diseases.

[0020] Furthermore, since Lactococcus lactis itself has probiotic potential, and the engineered strain of this invention can produce polypeptides with metabolic regulation functions in situ, this strain can also be developed into a compound live bacteria preparation with both microecological regulation and active molecule delivery functions, which has application prospects in the field of metabolic health.

[0021] Compared with the prior art, the present invention has the following beneficial effects: (1) By performing three site-directed mutations on Exendin-4: mutating asparagine at position 28 to glutamine to enhance chemical stability, and eliminating the Lys12-Arg13 trypsin cleavage site to resist intestinal degradation, thereby improving its stability in the gastrointestinal environment and oral bioavailability, the core obstacle to oral application of Exendin-4 is effectively overcome.

[0022] (2) The food-grade host lactococcus NZ3900 and NICE induction system, which are recognized as safe (GRAS), are used, and combined with the antibiotic-free lacF functional complementarity screening strategy. The entire production system is safe, environmentally friendly and meets the requirements of food / drug supervision. Compared with traditional expression systems such as Escherichia coli, this invention can achieve efficient extracellular secretion of the target protein and avoid inclusion body formation and complex in vitro refolding process.

[0023] (3) The fermentation method provided by the present invention has a clear culture medium composition, low cost, clear and controllable process parameters, and is easy to scale up production, thus having significant industrial advantages.

[0024] (4) Using engineered probiotics themselves as oral delivery carriers for live bacteria, producing and releasing modified Exendin-4 in situ in the intestine, realizing integrated production-delivery-treatment, and providing a new technology platform for the development of next-generation oral peptide drugs. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The image shows the pNZ8149-UEm-His recombinant plasmid.

[0027] Figure 2 The image shows the secretion and expression results of the target protein in the recombinant engineered bacteria pNZ8149-UEm-His.

[0028] Figure 3 The growth curve and target protein expression results of the recombinant engineered strain NZ3900 / pNZ8149-UEm-His are shown in the figure. Detailed Implementation

[0029] To make the technical problem to be solved, the technical solution and the beneficial effects of the present invention clearer, the following description is provided in conjunction with the appendix. Figure 1-3 The present invention will be further described in detail with reference to Examples 1-4.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Example 1: Construction of the pNZ8149-UEm-His recombinant plasmid (1) Obtaining the modified Exendin-4 gene fragment The nucleotide sequence of the triple-modified Exendin-4 described in this invention was synthesized artificially by a specialized institution, as shown in SEQ ID NO:1. Using this synthesized fragment as a template, high-fidelity PCR was performed using the primer pair shown in Table 1 (Exendin-4-F / Exendin-4-R). The PCR amplification program was as follows: 98℃ pre-denaturation for 2 min; followed by 30 cycles (98℃ denaturation for 10 s, 65℃ annealing for 30 s, 72℃ extension for 60 s); and finally, 72℃ extension for 5 min. After the reaction, the product was subjected to agarose gel electrophoresis, and purified using a DNA gel extraction kit to obtain the target gene fragment. This fragment contains a His tag coding sequence and is named the UEm-His fragment (abbreviated as UEm fragment).

[0032] Table 1 PCR reaction system (2) Linearization preparation of expression vector pNZ8149 The food-grade vector plasmid pNZ8149 and the host bacterium *Lactococcus lactis* NZ3900 used for expression were purchased commercially. The vector plasmid pNZ8149 was extracted in large quantities using an alkaline lysis method.

[0033] (3) Construction of pNZ8149-UEm-His recombinant plasmid Recombinant plasmids were constructed using the Kinmen cloning method. The UEm gene fragment purified in step (1) was mixed with the linearized pNZ8149 vector obtained in step (2) according to the system shown in Table 3.

[0034] Table 2 Primer sequences used for strain construction Table 3 Kinmen cloning reaction system Both DNA fragments were cleaved using Bsa I-HF v2 restriction endonuclease (recognition site: 5'-GGTCTC-3'). Since the recognition sequence of this enzyme was pre-designed on the primers of both the target fragment and the vector (sequences shown in Table 2), complementary 4 bp sticky ends were generated after cleavage. Subsequently, a one-step enzyme digestion-ligation reaction was performed at 16°C for 4 hours under the catalysis of T4 DNA ligase, achieving seamless and directional assembly of the DNA fragments. The reaction system is shown in Table 3. After the reaction, the ligation product was purified and concentrated using ethanol precipitation and dissolved in 10 μL of sterile water for later use. A schematic diagram of the recombinant plasmid is shown in [Table 3]. Figure 1 .

[0035] (4) Validation of recombinant plasmids The purified ligation product was transformed into competent *E. coli* cells, plated on LB agar plates containing antibiotics, and incubated overnight at 37°C. Single clones were picked for colony PCR identification. Positive clones were expanded and cultured, and plasmids were extracted and sent to a sequencing company for full-length sequencing. Sequencing results confirmed that the inserted sequence of the obtained plasmid was completely identical to the designed SEQ ID NO:1, indicating that the recombinant plasmid was successfully constructed and named pNZ8149-UEm-His.

[0036] Example 2: Construction of recombinant Lactococcus lactis engineered strain NZ3900 / pNZ8149-UEm-His To obtain recombinant lactococci capable of expressing modified Exendin-4, this embodiment transforms the recombinant plasmid pNZ8149-UEm-His constructed and verified in Example 1 into food-grade host bacteria NZ3900 via electroporation. The specific steps are as follows.

[0037] (1) Preparation of Lactococcus lactis NZ3900 electrocompetent cells The preserved Lactococcus lactis NZ3900 glycerol strain was streaked onto GM17 solid medium and incubated at 30°C for 24 hours.

[0038] The GM17 culture medium formula is as follows: glucose 0.5%, soybean peptone 5%, peptone 2.5%, casein peptone 2.5%, yeast extract 2.5%, beef extract 5%, lactose 5%, sodium ascorbate 0.5%, sodium β-glycerophosphate 19%, and magnesium sulfate 0.25%.

[0039] A single colony was picked and inoculated into 5 mL of liquid GM17 medium, and incubated overnight at 30°C as a primary seed culture. This seed culture was then transferred to 40 mL of fresh GM17 medium at a 5% inoculation rate and incubated at 30°C until OD500 was reached. 600 The value is approximately 0.4. All subsequent operations were performed on ice or at 4°C.

[0040] Collect the bacterial cells by centrifugation at 4°C and 6000×g for 5 minutes. Wash and resuspend the cells sequentially with the following pre-cooled washing buffer: Washing solution I (40 mL): contains 0.5 M sucrose, 20 mM MgCl2, and 10% (v / v) glycerol; Washing solution II (20 mL): containing 0.5 M sucrose, 1 mM EDTA, and 10% (v / v) glycerol, incubated on ice for 15 minutes; Washing solution III (10 mL): contains 0.5 M sucrose and 10% (v / v) glycerol.

[0041] After each resuspending, centrifuge at 4°C and 6000×g for 10 minutes, and discard the supernatant. Finally, gently resuspend the bacterial pellet with 400 μL of washing buffer III, aliquot 45 μL into pre-chilled centrifuge tubes, and immediately store at -80°C for later use.

[0042] (2) Electroporation conversion Take one tube of prepared NZ3900 competent cells and thaw on ice. Add 5 μL of the recombinant plasmid pNZ8149-UEm-His DNA (approximately 100 ng / μL) constructed and verified correctly in Example 1, mix gently, and transfer to a pre-chilled 0.2 cm electroporation cuvette. Incubate on ice for 10 minutes. Blot dry the outer wall of the electroporation cuvette with filter paper and perform electroporation using an electroporator at the following parameters: voltage 2.5 kV, capacitance 25 μF, resistance 200 Ω, and pulse duration typically 4.5–6.0 ms.

[0043] Immediately after the electroporation, add 200 μL of pre-recovery medium (GM17 basal medium supplemented with 0.5 M sucrose, 20 mM MgCl2, and 2 mM CaCl2) to the cup and gently mix with a pipette. After incubating on ice for 10 minutes, transfer all the bacterial culture to a centrifuge tube containing 1.5 mL of the above recovery medium and incubate at 30°C for 2 hours to complete the recovery process.

[0044] (3) Antibiotic-free screening based on lacF complementarity Take 200-500 μL of revived bacterial culture and spread it evenly on an Ellike antibiotic-free selection plate. The Elliker selection medium formula is as follows: 1 g tryptone, 0.67 g yeast nitrogen source (YNB), 0.4 g NaCl, 0.15 g sodium acetate, 0.05 g sodium ascorbate, 0.5 g lactose, 1.5 g agar, add ddH2O to 99 mL, adjust the pH to 7.5 with NaOH, sterilize, cool to about 60°C, add 1 mL of 0.4% (w / v) bromocresol purple solution, mix well, and pour the plate.

[0045] The plates were incubated at 30°C for 48 hours. Because the lacF gene on the recombinant plasmid encodes lactose permease, compensating for the deficiency of the lacF gene on the host NZ3900 chromosome, positive transformants were able to utilize lactose to produce acid, causing the bromocresol purple indicator around the colonies to change from purple to yellow. Single yellow colonies were selected for further verification.

[0046] (4) Molecular verification of recombinant engineered bacteria Pick the above-mentioned yellow single colonies and inoculate them into 5 mL of LLB liquid medium (containing 1% peptone, yeast extract, soybean peptone, and lactose), and incubate them statically at 30°C overnight. Extract bacterial plasmids using a plasmid extraction kit. Using these plasmids as templates, perform colony PCR verification using primers UEm-F / UEm-R. Clones that amplify the expected size band are considered preliminary positive clones.

[0047] The clones that initially tested positive were sent to a commercial sequencing company for DNA sequencing. Sequencing comparison showed that the modified Exendin-4 coding sequence carried in the plasmid was completely consistent with the designed SEQ ID NO:1.

[0048] The correctly sequenced strain was expanded and preserved, thus becoming the recombinant Lactococcus lactis engineered strain constructed in this invention, named NZ3900 / pNZ8149-UEm-His. This strain is the recombinant Lactococcus lactis engineered strain described in claim 1.

[0049] Example 3: Method for modifying Exendin-4 by fermentation production of recombinant engineered bacteria (1) Seed liquid preparation Take 1 μL of bacterial culture from the glycerol culture tube of the engineered strain NZ3900 / pNZ8149-UEm-His constructed and verified in Example 2, and inoculate it into a 250 mL shake flask containing 50 mL of LLB-whey medium. Incubate at 30°C for 12 hours until the bacterial growth enters the late logarithmic growth phase (OD2). 600 (≈0.42), used as primary seed liquid.

[0050] (2) Fermentation medium The LLB-whey culture medium used in this embodiment is prepared as follows: Solution A: Dissolve 50 g of whey powder (approximately 70% lactose content) in ultrapure water and bring the volume to 1 L. After stirring thoroughly, adjust the pH to 5.5 with 6 mol / L HCl and sterilize at 115°C for 30 minutes. After cooling to room temperature, centrifuge at 4000×g for 30 minutes and discard the precipitate. Take the supernatant, adjust the pH to 6.8 with 6 mol / L NaOH, and sterilize again at 115°C for 30 minutes. After cooling, centrifuge as above and discard the precipitate. Filter the supernatant through a 0.45 μm filter membrane for sterilization and store at 4°C for later use.

[0051] Solution B: Weigh 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, and 2.5 g of lactose. Add 25 mL of 1 M sodium phosphate buffer (pH 7.2), dissolve in ultrapure water and adjust the pH to 7.2. Finally, bring the volume to 500 mL and sterilize for later use.

[0052] Before use, mix equal volumes of solution A and solution B to obtain LLB-whey medium.

[0053] (3) Fermentation tank inoculation and culture The above-mentioned primary seed culture was transferred at an inoculum rate of 10% (v / v) to a 2 L fermenter containing 1000 mL of LLB-whey medium. Fermentation control parameters were set as follows: temperature 30°C, stirring speed 300 rpm, and aeration rate 1.5 vvm (volume / volume / min). After the temperature inside the fermenter stabilized, 1.5 mL of polyether defoamer was added. The pH of the fermentation broth was precisely controlled and stabilized at 7.0 by automatically adding 2 M NaOH or 2 M HCl solution.

[0054] (4) Induced expression and process control Real-time monitoring of fermentation broth OD 600 When OD600 reaches 0.35±0.03, a filtered and sterilized nisin A solution is added to the fermenter to achieve a final concentration of 10 ng / mL in the culture medium, thereby initiating the induced expression of the target protein.

[0055] Simultaneously with induction, a feed solution was started at a constant flow rate of 20 mL / h. The feed solution was an equal volume mixture of 50% (w / v) lactose solution and 10% (w / v) peptone-yeast extract (1:1) mixture to provide carbon and nitrogen sources for continuous cell growth and protein synthesis.

[0056] (5) Fermentation termination and product detection Fermentation was terminated after 36 hours of continuous fermentation induced by Nisin A. A sample of the fermentation broth was taken and centrifuged at 4°C and 6000×g for 10 minutes to obtain the fermentation supernatant containing the secreted protein.

[0057] The modified Exendin-4 in the fermentation supernatant was quantitatively detected using the detection method described in Example 4 (such as the His-Tag ELISA kit). Experimental results showed that the modified Exendin-4 was successfully secreted extracellularly, with a final yield of 8.35 ± 0.01 mg / L in the supernatant. This result demonstrates that the engineered bacteria and fermentation process constructed in this invention can efficiently achieve the secretory expression of the target protein.

[0058] Example 4 Detection and analysis of modified Exendin-4 in fermentation products To verify the secretion expression level and kinetics of the target protein in the recombinant engineered strain NZ3900 / pNZ8149-UEm-His during fermentation, the fermentation broth samples collected in Example 3 were tested as follows.

[0059] (1) Sample pretreatment and protein concentration Take 50 mL of fermentation broth at different time points after fermentation induction (e.g., 0, 2, 4, 6, 8, 12, 24, and 36 h) and place them on ice immediately. Centrifuge the samples at 4 °C and 6000 × g for 10 minutes, carefully separate and collect the supernatant.

[0060] Secretory proteins in the supernatant were concentrated using the trichloroacetic acid-acetone precipitation method: 1% (by volume) of 4% sodium deoxycholate solution was added to the supernatant, mixed, and incubated at 4°C for 1 hour. Then, 10% (by volume) of 100% (w / v) trichloroacetic acid solution was added, mixed, and incubated at -20°C for at least 1 hour to allow for complete protein denaturation and precipitation. The precipitate was centrifuged at 12000×g for 15 minutes at 4°C, and the supernatant was carefully discarded. The protein precipitate was washed three times with 5 mL of pre-chilled acetone, centrifuged after each wash under the same conditions. The precipitate was dried in a ventilated area or on ice, and finally resuspended in 1 / 10 of the original supernatant volume of pH 7.0 phosphate buffer to obtain a concentrated protein sample for Western blotting analysis. For quantitative detection, fresh supernatant after centrifugation was used directly.

[0061] (2) Western Blot detection of expression Take 30 μL of the above concentrated protein sample and mix it with 120 μL of 5×SDS loading buffer. Boil in a 100℃ metal bath for 5 minutes. Take 15-20 μL of the sample for Tricine-SDS-PAGE electrophoresis (using a 16% separating gel). After electrophoresis, transfer the protein to a PVDF membrane. Block with TBST solution containing 5% skim milk for 1 hour, then incubate overnight at 4℃ with mouse anti-His-tagged monoclonal antibody (1:2000 dilution). After washing, incubate at room temperature for 1 hour with horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:5000 dilution). After development with ECL chemiluminescent substrate, acquire the signal using a chemiluminescence imaging system. The results are as follows. Figure 2As shown, the successful secretory expression of the modified Exendin-4 is confirmed. Lane 1, representing NZ3900 / pNZ8149 (empty vector transformed into NZ3900), showed no specific band for the target protein, indicating that the empty vector-transformed bacteria did not express the target protein under the same conditions. Lanes 2-9 correspond to recombinant engineered bacterial samples fermented for 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, and 16 h after adding the inducer Nisin, respectively.

[0062] (3) Quantitative analysis by enzyme-linked immunosorbent assay Commercially available His-Tag ELISA kits were used to accurately quantify the target protein in the fermentation supernatant. Fermentation supernatants collected at different time points were appropriately diluted, and the procedure was strictly followed according to the kit instructions. Purified recombinant Exendin-4 (UEm-His) was used as a standard to plot a standard curve, and the coefficient of determination R of the standard curve was required to be measured. 2 >0.99. Calculate the concentration (mg / L) of modified Exendin-4 in the supernatant at each time point based on the standard curve.

[0063] Quantitative results such as Figure 3 As shown, A represents the growth curve; B represents the content of the target protein. Data shows that after Nisin A induction, the target protein begins to be secreted and gradually accumulates, reaching a plateau phase after approximately 24-36 hours of fermentation, with a maximum yield of 8.35 mg / L. This result is consistent with the cell growth curve (OD). 600 The correlation indicates that the engineered bacteria and fermentation process described in this invention can achieve efficient and stable secretion production of modified Exendin-4.

[0064] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents. SEQ ID No: 1

Claims

1. A recombinant Lactococcus lactis engineered strain expressing modified Exendin-4, characterized in that, The engineered bacteria were constructed by cloning the modified Exendin-4 encoding gene into the food-grade expression plasmid pNZ8149 to obtain the recombinant plasmid pNZ8149-UEm-His, and then introducing this recombinant plasmid into Lactococcus lactis NZ3900. The modified Exendin-4 encoding gene contains the following three site-directed mutations: (1) Asparagine at position 28 is mutated to glutamine; (2) Eliminate the Lys12-Arg13 trypsin-specific recognition site through point mutation; (3) A cysteine ​​residue is added to the C-terminus; the genotype of the engineered bacteria is NZ3900 / pNZ8149-UEm-His.

2. The recombinant Lactococcus lactis engineered strain according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the modified Exendin-4 is shown in SEQ ID No:

1.

3. The recombinant lactococcus engineered strain according to claim 1 or 2, characterized in that, The recombinant plasmid pNZ8149-UEm-His contains the PnisA promoter, the lacF selection marker gene, and the replication initiation proteins RepA and RepC.

4. A method for constructing the recombinant Lactococcus lactis engineered strain as described in claim 1, characterized in that, Includes the following steps: (1) Synthesize the gene fragment encoding the modified Exendin-4; (2) The gene fragment was cloned into the food-grade expression plasmid pNZ8149 to obtain the recombinant plasmid pNZ8149-UEm-His; (3) The recombinant plasmid was transformed into Lactococcus lactis NZ3900 competent cells to obtain the engineered bacteria.

5. The construction method according to claim 4, characterized in that, In step (2), the cloning is performed by using the Jinmen cloning method to obtain the recombinant plasmid.

6. The construction method according to claim 4, characterized in that, In step (3), the conversion involves introducing the recombinant plasmid into competent cells via electroporation and screening for positive clones on antibiotic-free lactose medium using lacF gene functional complementation.

7. A method for producing modified Exendin-4, characterized in that, include: The recombinant lactococcus engineered strain according to any one of claims 1 to 3 is fermented to obtain a culture containing the recombinant lactococcus engineered strain, and the modified Exendin-4 is isolated or purified from the culture.

8. The production method according to claim 7, characterized in that, The fermentation was carried out in LLB-whey medium, when the bacterial cell OD... 600 When the concentration reaches 0.3-0.4, Nisin A is added to a final concentration of 5-15 ng / mL for induction.

9. The production method according to claim 7 or 8, characterized in that, The modified Exendin-4 is secreted extracellularly and obtained by collecting the supernatant through centrifugation or filtration.

10. The recombinant Lactococcus lactis engineered strain according to any one of claims 1 to 3, characterized in that, The engineered bacteria can secrete and express the modified Exendin-4 protein under induction conditions.

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