Engineering probiotics secreting CXCL12 as well as construction method and application thereof

Abstract

The invention provides engineering probiotics capable of secreting CXCL12 as well as a construction method and application of the engineering probiotics, and relates to the technical field of biology. A carrier containing a P32 (pgm) promoter fragment, a 1p3050 secretory signal peptide and a CXCL12 gene is constructed and transferred into a host bacterium lactobacillus plantarum WCFS1, an efficient secretory expression system of the engineering probiotics of the CXCL12 is provided, the secretory expression quantity of the CXCL12 is increased to 13.40 + / -0.20 ng / mu L, the engineering probiotics can accelerate healing of chronic wound surfaces of diabetes mellitus, and the CXCL12 can be used for treating chronic wound surfaces of diabetes mellitus. And a new thought and technical support are provided for clinical treatment of diabetic chronic wounds in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an engineered probiotic that secretes CXCL12 and a construction method and application thereof. Background Art

[0002] Chronic diabetic wounds are difficult to heal due to their complex pathological basis, resulting in patients' mobility problems, amputation and even death, which seriously affects the patients' quality of life and mental health, and imposes a heavy burden on social economy and medical resources. According to relevant statistics from the International Diabetes Federation (IDF), the number of adult diabetic patients in the world reached 537 million in 2021, resulting in an estimated global health expenditure of US$966 billion; it is estimated that by 2030 and 2045, the number of adult diabetic patients in the world will reach 643 million and 783 million. Due to the lack of effective prevention and control measures, the sharp increase in chronic wounds and foot ulcer complications caused by the sharp rise in the incidence of diabetes has become an increasingly serious clinical problem. Due to the long-term chronic inflammation, metabolic abnormalities, difficulty in healing or non-healing of chronic diabetic wounds (skin wounds and foot ulcers), conventional treatments such as debridement, skin transplantation and systemic antibiotic use cannot achieve satisfactory results.

[0003] In recent years, the rapid development of synthetic biology has enabled us to design complex genetic circuits in model bacteria to transform the host genome, construct corresponding genetically engineered bacteria and endow them with new functions for targeted regulation or treatment of diseases, which has great application potential and development value. In summary, an engineered probiotic can be constructed for wound healing in diabetes. Summary of the invention

[0004] The present invention aims to provide an engineered probiotic that secretes CXCL12, and a construction method and application thereof. Lactobacillus plantarum WCFS1 is used as a host bacterium, and a set of strong constitutive promoter P based on the strain is identified and characterized by using the principle of synthetic biology technology. 32(pgm) and lp_3050 secretion signal peptide.

[0005] The first object of the present invention is to provide an engineered probiotic that secretes CXCL12, including constitutive P 32(pgm) Promoter, exotransglycosylation secretion signal peptide lp_3050 and CXCL12 gene.

[0006] Optionally, the constitutive P 32(pgm) The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0007] Optionally, the constitutive P 32(pgm) The promoter includes P 32Promoter and pgm promoter ribosome binding site RBS.

[0008] Optionally, the nucleotide sequence of the extracellular transglycosylation enzyme secretion signal peptide lp_3050 is shown as SEQ ID NO.2.

[0009] Optionally, the nucleotide sequence of the CXCL12 gene is shown in SEQ ID NO.3.

[0010] Optionally, the host bacteria of the engineered probiotics is Lactobacillus plantarum WCSF1.

[0011] The second object of the present invention is to provide a recombinant protein delivery system, comprising a recombinant vector, an extracellular transglycosylation enzyme secretion signal peptide lp_3050 and a CXCL12 gene.

[0012] The recombinant protein delivery system provided by the present invention is 32 Compared with the commonly used usp45 signal peptide combination, the ability of the promoter to secrete and express the gusA reporter gene was increased by nearly 5 times, and the secretory expression level of CXCL12 could reach 13.40±0.20ng / μL.

[0013] Optionally, the recombinant vector includes pTRK892-P 32(pgm) Vector and pHSpBR256-P 32(pgm) Any one of the vectors.

[0014] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0015] The applicant shall submit P 32(pgm) The promoter fragment, lp_3050 secretion signal peptide and CXCL12 gene were expressed in pTRK892-P 32(pgm) Vector or pHSpBR256-P 32(pgm) The vector was assembled to construct a high-efficiency secretory expression system of the CXCL12 gene, and it was transferred into the host bacteria WCFS1 Lactobacillus plantarum.

[0016] The qualitative and quantitative results of GusA enzyme activity proved that in Lactobacillus plantarum WCFS1, the secretion ability of the heterologous protein of the lp_3050 secretion signal peptide was significantly stronger than the commonly used usp45 signal peptide; at the same time, Western-Blot detection found that the CXCL12 gene can be stably secreted and expressed in WCFS1. Therefore, it can be considered that the engineered Lactobacillus plantarum for delivering chemokine CXCL12 was successfully constructed.

[0017] The third object of the present invention is to provide an application of an engineered probiotic in the healing of chronic diabetic wounds, the management of diabetic foot ulcers and the healing of surgical wounds.

[0018] The engineered probiotics secreting CXCL12 provided by the present invention can effectively promote the healing of chronic wounds in mice, and provide new ideas and technical support for the clinical treatment of chronic diabetic wounds in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly introduce the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments.

[0020] Figure 1 pTRK898-P constructed in Example 1 of the present invention 32(pgm) Plasmid map of the vector;

[0021] Figure 2 pTRK898-P constructed in Example 1 of the present invention 32(pgm) -SP lp_3050 Plasmid map of the vector;

[0022] Figure 3 pTRK898-P constructed in Example 1 of the present invention 32(pgm) -SP usp45 Plasmid map of the vector;

[0023] Figure 4 pHSpBR256-P constructed in Example 1 of the present invention 32(pgm) Plasmid map of the vector;

[0024] Figure 5 pHSpBR256-P constructed in Example 1 of the present invention 32(pgm) -SP lp_3050 -Plasmid map of CXCL12His vector;

[0025] Figure 6 pHSpBR256-P constructed in Example 1 of the present invention 32(pgm) -SP lp_3050 -Plasmid map of CXCL12HiBit vector;

[0026] Figure 7 To identify the GusA enzyme activity of the secretion system of Lactobacillus plantarum WCFS1 mediated by the lp_3050 secretion signal peptide in the present invention;

[0027] Figure 8 The present invention is to construct and Western-Blot detect the engineered probiotic bacteria that secrete and express CXCL12;

[0028] Fig. 9 This is a graph showing the effects of different engineered probiotics on the treatment of chronic diabetic wounds for 0 to 12 days;

[0029] Fig.10 The wound healing rates of chronic diabetic wounds treated with different engineered probiotics in the present invention for 3 to 12 days. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described through the following embodiments in conjunction with the accompanying drawings. However, the following embodiments are illustrative and are only used to specifically describe the present invention rather than to limit the present invention.

[0031] The present invention provides an engineered probiotic that secretes CXCL12, including a constitutive P 32(pgm) Promoter, exotransglycosylation secretion signal peptide lp_3050 and CXCL12 gene.

[0032] In some embodiments, the constitutive P 32(pgm) The nucleotide sequence of the promoter is shown in SEQ ID NO.1.

[0033] In some embodiments, the constitutive P 32(pgm) The promoter includes P 32 Promoter and pgm promoter ribosome binding site RBS.

[0034] In some embodiments, the nucleotide sequence of the extracellular transglycosylation enzyme secretion signal peptide lp_3050 is shown as SEQ ID NO.2.

[0035] In some embodiments, the nucleotide sequence of the CXCL12 gene is shown as SEQ ID NO.3.

[0036] In some embodiments, the host bacteria of the engineered probiotics is Lactobacillus plantarum WCSF1.

[0037] The present invention also provides a recombinant protein delivery system, comprising a recombinant vector, an extracellular transglycosylation enzyme secretion signal peptide lp_3050 and a CXCL12 gene.

[0038] In some embodiments, the recombinant vector comprises pTRK892-P 32(pgm) Vector and pHSpBR256-P 32(pgm) Any one of the vectors.

[0039] The present invention also provides a method for constructing a secretory expression system of a CXCL12 probiotic, including Gibson assembly of an amplified fragment and a linearized vector, preparation of competent Escherichia coli cells and plasmid extraction, and preparation of competent Lactobacillus plantarum cells and electrotransformation.

[0040] In some embodiments, the secretory expression system includes P 32Promoter fragment, lp_3050 secretion signal peptide, CXCL12 gene, pTRK892-P 32(pgm) Vector or pHSpBR256-P 32(pgm) Vector and host bacteria Lactobacillus plantarum WCFS1.

[0041] The present invention also provides an application of the engineered probiotics in the healing of chronic diabetic wounds, the management of diabetic foot ulcers and the healing of surgical wounds.

[0042] The pTRK892 plasmid used in the embodiments of the present invention was purchased from Addgene, the pHSpBR256 plasmid was constructed by Haosheng Shen et al. from the National University of Singapore, and the remaining plasmids were constructed in this study; the LB (Luria-Bertani) medium used in the embodiments of the present invention contains 200 μg / mL erythromycin and 100 μg / mL ampicillin, and the MRS (DeMan, Rogosa and Sharpe) medium contains 10 μg / mL erythromycin; the materials, reagents, etc. used in the embodiments of the present invention, unless otherwise specified, can be obtained from commercial channels, and the experimental methods for which specific conditions are not specified in the embodiments are all in accordance with conventional conditions or the conditions recommended by the manufacturer.

[0043] Example 1

[0044] Example 1 of the present invention provides a method for constructing a CXCL12 engineered probiotic secretory expression system, which specifically includes: Gibson assembly of amplified fragments and linearized vectors, preparation of Escherichia coli competent cells, and preparation of Lactobacillus plantarum competent cells and electroporation.

[0045] The present invention uses Snapgene software (version 6.0.2, www.snapgene.com) to simulate the plasmid to be constructed and design the corresponding primer sequences. The primer annealing temperature is controlled at 55° C.-60° C. The primer sequences used are shown in Table 1.

[0046] Table 1 Primers used in plasmid construction of the present invention

[0047]

[0048]

[0049]

[0050] 1. Gibson assembly of amplified fragments, linearized vectors and preparation of competent E. coli cells

[0051] S1, P 32The promoter fragment (amplification primers: SEQ ID NO: 4 and SEQ ID NO: 5) was assembled with the pTRK892 vector plasmid double-digested with NcoI and EcoRI, and the recombinant product was transformed into E. coli competent cells to construct pTRK892-P 32(pgm) Carrier ( Figure 1 ), the sequencing primer is SEQ ID NO: 21;

[0052] S2, the N-terminal fragments of the gusA reporter gene (primers: SEQ ID NO: 9 / SEQ ID NO: 14 and SEQ ID NO: 10) were fused with the lp_3050 secretion signal peptide (amplification primers: SEQ ID NO: 6 and SEQ ID NO: 7; fusion primers: SEQ ID NO: 11 and SEQ ID NO: 10) and the usp45 signal peptide (amplification primers: SEQ ID NO: 12 and SEQ ID NO: 13; fusion primers: SEQ ID NO: 11 and SEQ ID NO: 10) and EcoRI and NotI double-digested pTRK892-P 32(pgm) The vector plasmid was assembled and the recombinant product was transferred into E. coli competent cells to construct pTRK892-P 32(pgm) -SP lp_3050 Carrier ( Figure 2 ) and pTRK892-P 32(pgm) -SP usp45 Carrier ( Figure 3 ), the sequencing primers are SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25;

[0053] S3, P 32 The promoter fragment was assembled with the pHSpBR256 vector plasmid double-digested with NcoI and EcoRI, and the recombinant product was transformed into E. coli competent cells to construct pHSpBR256-P 32(pgm) Carrier ( Figure 4 ), the sequencing primer is SEQ ID NO: 26;

[0054] S4, the lp_3050 secretion signal peptide was fused to the CXCL12 gene (amplification primers: SEQ ID NO: 15 and SEQ ID NO: 16; fusion primers: SEQ ID NO: 11 and SEQ ID NO: 16), the CXCL12 gene with a His tag at the C-terminus (amplification primers: SEQ ID NO: 15 and SEQ ID NO: 17; fusion primers: SEQ ID NO: 11 and SEQ ID NO: 18), and the CXCL12 gene with a HiBit tag at the C-terminus (amplification primers: SEQ ID NO: 15 and SEQ ID NO: 19; fusion primers SEQ ID NO: 11 and SEQ ID NO: 20) and pHSpBR256-P double-digested with EcoRI and PstI. 32(pgm) The vector was assembled and the recombinant product was transferred into E. coli competent cells to construct pHS256-P 32(pgm) -SP lp_3050 -CXCL12 vector, pHSpBR256-P 32(pgm) -SP lp_3050 -CXCL12His vector ( Figure 5 ) and pHSpBR256-P 32(pgm) -SP lp_3050 -CXCL12HiBit vector ( Figure 6 ), the sequencing primer is SEQ ID NO: 26;

[0055] The PCR reaction system during amplification was: 50 μL system, including 2 μL of 10 μM forward and reverse primers, 25 μL of 2× PrimeSTAR Premix, 2.5 μL of DNA template, and ddH2O to 50 μL; PCR reaction program: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 60°C annealing for 15 s, 72°C extension for 10 s, for a total of 25 cycles; 72°C final extension for 2 min; agarose gel electrophoresis (1% w / v) to separate the PCR amplification products, and the electrophoresis conditions were: 120V constant voltage, 30 min-50 min. After the electrophoresis, the PCR products were observed by gel imaging, the amplified products of the target fragments were cut out, and the Promega gel recovery kit ( After purification using SVGel and PCR Clean-Up System, Catalog No. A9282), the concentration was determined using Nanodrop 2000 and stored at -20°C for future use;

[0056] S5. Use NEB's Gibson assembly reagent (NEBuilder HiFi DNA Assembly Master Mix, Catalog No. E2621) to assemble the amplified fragment and the linearized vector plasmid. The PCR reaction system is: 10 μL system, including 50 ng of linearized plasmid, 5 μL of 2× Assembly Master Mix, 50 ng of inserted fragment, and ddH2O to make up to 10 μL; PCR reaction procedure: incubate at 50°C for 30 min.

[0057] S6. The transformation of E. coli competent cells includes: transferring the activated E. coli into E. coli Top10 competent cells, inoculating 1% of the E. coli into 100 mL of LB liquid culture medium, and culturing at 37°C and 200 rpm for 2 h until the OD 600 0.5-0.6, stand on ice for 10 minutes, centrifuge at 4°C, 3000rpm for 5 minutes, collect the bacterial precipitate; take 30mL of pre-cooled 60mM CaCl2 solution and blow the bacterial precipitate twice, centrifuge at 4°C, 3000rpm for 5 minutes, collect the bacterial precipitate; add 1mL of pre-cooled 60mM CaCl2 + 15% glycerol solution, resuspend and mix the bacterial precipitate, package in 100 μL / tube, and store at -80°C for later use; take out the competent cells and thaw them on ice, add the above-mentioned recombinant products respectively, and ice bath for 30 minutes; heat shock at 42°C for 60 seconds and quickly put them on ice for 3 minutes; add 900 μL of LB liquid culture medium and shake and culture at 37°C and 200 rpm for 1 hour; spread on LB plate; incubate the plate upside down at 37°C incubator for 10 hours, observe the growth of the colonies, pick several single colonies for PCR identification, expand the culture of the successfully identified single colonies, use Qiagen plasmid miniprep kit (Plasmid miniprep kit, product number 16181) to extract the plasmid, and send it to a sequencing company for plasmid sequencing.

[0058] 2. Preparation of competent cells of Lactobacillus plantarum and electroporation

[0059] The activated WCFS1 Lactobacillus plantarum was inoculated into 100 mL MRS + 2% glycine liquid medium at 1% by volume and cultured at 30°C until OD 6000.5-0.6, stand on ice for 15 minutes, centrifuge at 4°C, 3000r / min for 5 minutes to collect bacterial precipitates; take 30mL of pre-cooled 0.1mM MgCl2 solution to blow the bacterial precipitate twice, centrifuge at 4°C, 3000r / min for 5 minutes to collect bacterial precipitates; take 30mL of pre-cooled 30% PEG1500 to blow the bacterial precipitate twice, centrifuge at 4°C, 5000rpm, 5min to collect bacterial precipitates; add 1mL of pre-cooled 30% PEG1500 solution to the centrifuge tube, resuspend and mix the bacterial precipitate, dispense 100μL / tube, and store at -80°C for use; Take competent cells and thaw them on ice, add 1.0 μg of the above-extracted plasmids, incubate on ice for 10 min, and then quickly transfer to a pre-cooled electroporation cup, and electrotransform for 10 ms under the conditions of voltage 2.0 kV, resistance 400 Ω, and electric pulse 25 μF. After electroporation, add 1 mL of MRS medium, incubate at 37 ° C for 2 h, then spread on an MRS solid culture dish, and invert and culture at 37 ° C for 2 days; pick a single clone and expand the culture in MRS liquid culture medium to prepare Lpw-SP lp_3050 -CXCL12-His and Lpw-SP lp_3050 -CXCL12-HiBit, Lpw-SP lp_3050 -CXCL12 engineered probiotic strains are available.

[0060] Characterization of the secretion system of Lactobacillus plantarum WCFS1

[0061] Using the gusA reporter gene, X-GluC and PNPG as effector substrates, the secretion expression ability of gusA mediated by the lp_3050 secretion signal peptide and the usp45 signal peptide was compared and analyzed. Figure 7 (A) shown.

[0062] Prepare an MRS plate containing 50 μg / mL X-Gluc chromogenic substrate (CAS: 114162-64-2) and take Lpw, Lpw-SP lp_3050 -gusA and Lpw-SP usp45 -gusA strain was streaked on a plate and inverted at 37°C. Figure 7 As shown in (B), compared with the wild-type Lactobacillus plantarum WCFS1 strain (Lpw), the engineered strain Lpw-SP lp_3050 -gusA showed obvious blue colonies and larger surrounding blue circles, while the engineered bacteria Lpw-SP usp45 The -gusA colonies and the blue circles around them are very weak.

[0063] The OD of each engineered bacteria after overnight culture was 600After adjusting to about 0.1, place it at the corresponding culture temperature and culture it for 24 hours. The OD of each strain was measured by an ELISA instrument. 600 ; 10 μL of culture medium was added to a 96-well plate containing 90 μL of gusA reaction buffer, and 2.5 μL of 0.1% SDS and 5 μL of chloroform were added, and the reaction was carried out at 37°C for 10 min; 20 μL of 50 mg / mL p-4-nitrophenyl-β-D-glucoside solution (PNPG, CAS: 10344-94-2) was added to the reaction system, and the mixture was gently mixed, and the reaction was carried out at 37°C for 30 min, and then 50 μL of 1 M sodium bicarbonate solution was added; after centrifugation to remove cell debris, 100 μL of supernatant was taken to a new 96-well plate, and the OD was measured. 405 Absorbance value. Miller units were used to quantitatively analyze GusA enzyme activity, and the calculation formula was as follows:

[0064]

[0065] like Figure 7 (C) shows that the genetically engineered bacteria Lpw-SP lp_3050 -gusA fermentation supernatant had significantly higher GusA activity than that of the engineered strain Lpw-SP lp_3050 -gusA(41684.27±2622.51vs.8906.93±250.65).

[0066] The comprehensive qualitative and quantitative results demonstrated that in Lactobacillus plantarum WCFS1, the secretion ability of heterologous proteins with lp_3050 secretion signal peptide was significantly stronger than that of usp45 signal peptide.

[0067] Western-Blot detection

[0068] 1.5 g TCA was added to 10 mL of sterile fermentation liquid. After reacting at low temperature for 2 hours, the precipitate was collected by high-speed centrifugation, washed twice with pre-cooled acetone, and the total protein was obtained after drying, which was stored at -20°C for later use; the total protein was separated by 12% SDS-PAGE electrophoresis and transferred to a membrane; after transfer, the PVDF membrane was blocked in 5% skim milk prepared in 1×TBST at room temperature for 2 hours, and then washed repeatedly with 1×TBST for 3 times, each time for 5 minutes; incubated with His tag antibody diluted 1:1000 at room temperature for 1 hour, and washed repeatedly with 1×TBST for 3 times, each time for 5 minutes; developed and exposed with a color developing solution for detection.

[0069] Refer to Promega's HiBit label quantitative detection kit ( The instructions for use of the HiBiTExtracellular Detection System (Cat. No. N2420) were used to detect CXCL12 containing the HiBit tag in the fermentation supernatant, and a standard curve was established to quantitatively calculate the content of CXCL12 HiBit.

[0070] Western-Blot results showed that compared with Lpw ( Figure 8 (B) Lane 4), three randomly selected engineered probiotics Lpw-SP lp_3050 -CXCL12-His fermentation supernatant can detect obvious His tag signal ( Figure 8 (B) Lanes 1-3) indicate that CXCL12 can be stably secreted and expressed in this system.

[0071] The results of HiBit quantitative label detection are as follows Figure 8 (C), engineered probiotics Lpw-SP lp_3050 The content of CXCL12-HiBit in the fermentation supernatant of -CXCL12-HiBit was as high as 13.40±0.20ng / μL. These results indicate that the engineered Lactobacillus plantarum for delivering chemokine CXCL12 has been successfully constructed.

[0072] To avoid the influence of signal tags on CXCL12 activity, we removed the His and HiBit tags and constructed the CXCL12 engineered probiotic Lpw-SP lp_3050 -CXCL12 (Lpw-CXCL12) is used for subsequent chronic wound treatment.

[0073] The therapeutic effects of different engineered probiotics on chronic diabetic wounds

[0074] All animal experimental protocols and handling methods in this study have been reviewed and approved by the Experimental Animal Ethics Committee of the School of Medicine of Nanchang University and the Experimental Animal Science Center of Nanchang University, with approval number NCULAE-20221228057.

[0075] Twelve 7-week-old male C57BL / 6 mice were purchased from Nanjing Cores Animal Co., Ltd. After 1 week of adaptive feeding with normal feed, they were given a high-sugar and high-fat diet for 8 weeks. Three mice were randomly selected as the control group, and the remaining 9 mice were intraperitoneally injected with 50 mg / kg / d of streptozotocin (STZ) for 5 consecutive days; the fasting blood glucose of the mice was measured, and the T2DM mouse model was successfully established when the fasting blood glucose of the mice was ≥16.67 mM / L for 3 consecutive times.

[0076] All mice were anesthetized and their backs were cut open 1 cm 2The wound surface of mice was smeared with sterile saline. The wound surface of mice in the control group (C) was smeared with sterile saline. The mice in the experimental group were randomly divided into 3 groups, with 3 mice in each group. The wound surface of the model group (M) was smeared with sterile saline. The wound surface of the Lpw group (Lpw) was smeared with 1×10 8 1×10 CFU of Lactobacillus plantarum WCFS1 wild strain Lpw and Lpw-CXCL12 group were applied to the wound surface 8 CFU of CXCL12 engineered probiotic Lpw-SP lp_3050 -CXCL12.

[0077] Every 3 days, photos were taken to record the wound healing of each group of mice. Image J software was used to count the wound area of ​​each group of mice and calculate the wound healing rate. The calculation formula for the wound healing rate is as follows:

[0078]

[0079] The wound healing status of mice in each group was as follows Fig. 9 As shown in Figure 2, the wound healing rate of mice was Fig.10 As shown in Table 2, the results showed that the wound healing rate of mice in group M was significantly lower than that in group C, indicating that the wound healing ability of T2DM mice was significantly reduced, manifested as chronic wounds; compared with group M, although Lpw wild bacteria treatment could slightly promote the healing of chronic wounds, the effect was not significant; after treatment with Lpw-CXCL12 engineered probiotics, the healing rate of chronic wounds was significantly higher than that of group M and group Lpw; in particular, in the early stage of chronic wound healing (day 3), the wound healing rate of the Lpw-CXCL12 group was even higher than that of the C group.

[0080] Table 2 Wound healing rate of mice

[0081]

[0082] Although the embodiments of the present invention are described in detail above, it is obvious to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are within the scope and spirit of the present invention as described in the claims. Moreover, the present invention described herein may have other embodiments and may be implemented or realized in a variety of ways.

Claims

1. An engineered probiotic that secretes CXCL12, characterized in that: Including constitutive P 32(pgm) promoter, exotransglycosylation secretion signal peptide lp_3050 and CXCL12 gene, the constitutive P 32(pgm) The nucleotide sequence of the promoter is shown in SEQ ID NO.1, the nucleotide sequence of the extracellular transglycosylation enzyme secretion signal peptide lp_3050 is shown in SEQ ID NO.2, and the nucleotide sequence of the CXCL12 gene is shown in SEQ ID NO.

3.

2. The engineered probiotic according to claim 1, characterized in that: The constitutive P 32(pgm) The promoter includes P 32 Promoter and pgm promoter ribosome binding site RBS.

3. The engineered probiotic according to claim 1, characterized in that: The host bacterium is Lactobacillus plantarum WCSF1.

4. A recombinant protein delivery system, characterized in that: It includes a recombinant vector, an extracellular transglycosylation enzyme secretion signal peptide lp_3050 and a CXCL12 gene.

5. The recombinant protein delivery system according to claim 4, characterized in that: The recombinant vector includes pTRK892-P 32(pgm) Vector and pHSpBR256-P 32(pgm) Any one of the vectors.

6. Use of the engineered probiotic according to any one of claims 1 to 3, or the recombinant protein delivery system according to any one of claims 4 to 5, in enhancing the delivery of CXCL12.

7. Use of the engineered probiotic according to any one of claims 1 to 3, or the recombinant protein delivery system according to any one of claims 4 to 5, in improving CXCL12 expression.

8. Use of the engineered probiotics according to any one of claims 1 to 3, or the engineered probiotics prepared by the recombinant protein delivery system according to any one of claims 4 to 5 in the healing of chronic wounds of diabetes, the management of diabetic foot ulcers and the healing of surgical wounds.

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