Stable lactic acid bacteria secretory expression vector and construction method thereof

Abstract

The invention relates to the field of microorganisms, and particularly provides a stable lactic acid bacteria secretory expression vector and a construction method thereof. The vector comprises a replication system, a resistance screening system and a target gene expression system, wherein the replication system comprises a lactic acid bacteria replicon sequence and an escherichia coli replicon sequence; the resistance screening system comprises an ermB gene and a regulatory element thereof; the target gene expression system comprises a secretion signal peptide sequence, a cloning site and a terminator sequence. The invention constructs three vectors: a basic vector, a self-linking vector and a constitutive vector. Experimental results show that the vector system can be stably copied and expressed in escherichia coli and lactobacillus plantarum, and has good genetic stability. The method can also be used for constructing recombinant lactic acid bacteria and expressing exogenous genes. The shuttle expression vector provided by the invention has the characteristics of stable structure, high expression efficiency and the like, and provides a new tool for constructing engineering strains.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a stable lactic acid bacteria secretion expression vector and a construction method thereof. Background Art

[0002] As a new type of live carrier, recombinant lactic acid bacteria has broad application prospects in the fields of vaccine and drug delivery. Lactobacillus plantarum ) has become an important host strain for constructing recombinant lactic acid bacteria due to its high safety and strong colonization ability. At present, the construction of recombinant lactic acid bacteria mainly relies on the shuttle expression vector system. The main problems of existing lactic acid bacteria expression vectors are: (1) lack of efficient secretion expression system, resulting in low expression of exogenous proteins; (2) insufficient genetic stability, and easy loss of target genes after continuous subculture; (3) lack of versatility and difficulty in meeting different experimental requirements. In addition, the existing vectors often require multiple enzyme digestions and ligations during the construction process, which is cumbersome and inefficient. Therefore, it is of great significance to develop a lactic acid bacteria expression vector system with good stability, high expression efficiency and simple operation. This not only helps to improve the construction efficiency of recombinant lactic acid bacteria, but also provides an important tool for the development of new microecological preparations and vaccine vectors. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a new type of stable lactic acid bacteria secretion expression vector and its preparation method and application. The system has the following technical features: a basic vector containing a complete functional module is constructed, a dual-host replication system, a resistance screening system and a target gene expression system are integrated, and stable replication in Escherichia coli and lactic acid bacteria can be achieved; a self-ligating vector is developed based on the basic vector, the cloning step is simplified, and the recombination efficiency is improved; a constitutive vector is constructed by integrating the P32 promoter sequence, and the efficient expression of exogenous genes is achieved; on this basis, a labeling system and a verification system can also be introduced to facilitate protein expression and functional verification. The present invention uses seamless cloning technology to construct a vector system, which avoids the cumbersome steps of multiple enzyme cutting and connection in traditional methods, is easy to operate, and has significantly improved efficiency. The vector system can be widely used in the construction of recombinant lactic acid bacteria and the expression of exogenous genes, and provides an important tool for the development of new microecological preparations and vaccine vectors. Specifically, the present invention provides the following technical solutions.

[0004] In a first aspect of the present invention, a stable lactic acid bacteria secretory expression vector is provided, which comprises a replication system, a resistance screening system and a target gene expression system; wherein the replication system is a lactic acid bacteria-Escherichia coli dual-host replication system, comprising a lactic acid bacteria replicon sequence and an Escherichia coli replicon sequence; the resistance screening system comprises a screening marker gene and its regulatory elements; the target gene expression system comprises a secretion signal peptide sequence, a cloning site and a terminator sequence; wherein the stable lactic acid bacteria secretory expression vector is selected from any one of the following: (1) a basic vector comprising the replication system, the resistance screening system and the target gene expression system; (2) a self-ligating vector obtained by self-ligating the basic vector; and (3) a constitutive vector having a P32 promoter sequence additionally integrated on the basis of the self-ligating vector.

[0005] In some embodiments of the present invention, the replication system of the present invention is a lactic acid bacteria-Escherichia coli dual-host replication system, including: (1) Lactobacillus replicon sequence: using 256ori sequence to enable the vector to stably replicate in lactic acid bacteria; and (2) Escherichia coli replicon sequence: using pUC-ori sequence to ensure the replication function of the vector in Escherichia coli. The resistance screening system of the present invention includes: (1) Screening marker gene: using ermB gene to confer erythromycin resistance to the host; and (2) Regulatory element: including ermB promoter sequence, ermB leader peptide sequence and ermB terminator sequence to regulate the expression of ermB gene. The target gene expression system of the present invention includes: (1) Secretion signal peptide sequence: to guide the secretory expression of the target protein; (2) Cloning site: set Nco I and Bam HI restriction site to facilitate the insertion of the target gene; and (3) terminator sequence: the cbh terminator sequence is used to ensure the termination of transcription of the target gene.

[0006] In some embodiments of the present invention, the sequence involved in the aforementioned elements may be a nucleotide sequence as shown below or a functional equivalent or optimized variant thereof. Wherein, the nucleotide sequence of the secretion signal peptide is shown in SEQ ID NO: 1. In some embodiments of the present invention, the nucleotide sequence of the lactic acid bacteria replicon is shown in SEQ ID NO: 2 (256ori sequence). In some embodiments of the present invention, the nucleotide sequence of the Escherichia coli replicon is shown in SEQ ID NO: 3 (pUC-ori sequence). In some embodiments of the present invention, the nucleotide sequence of the ermB gene is shown in SEQ ID NO: 4. In some embodiments of the present invention, the nucleotide sequence of the ermB promoter is shown in SEQ ID NO: 5. In some embodiments of the present invention, the nucleotide sequence of the ermB leader peptide is shown in SEQ ID NO: 6. In some embodiments of the present invention, the nucleotide sequence of the ermB terminator is shown in SEQ ID NO: 7. In some embodiments of the present invention, the nucleotide sequence of the cbh terminator is shown in SEQ ID NO: 8. In some embodiments of the present invention, when the stable lactic acid bacteria secretory expression vector is the constitutive vector, the nucleotide sequence of the P32 promoter is as shown in SEQ ID NO: 9.

[0007] In addition, the stable lactic acid bacteria secretory expression vector of the present invention may also include at least one of the following functional modules: a labeling system: for example, using a 3xFLAG tag to facilitate the detection of the expression and localization of the target protein; and a verification system: for example, including an SP6 promoter and an EGFP gene to verify the function and expression efficiency of the vector.

[0008] In some embodiments of the present invention, the sequences involved in the aforementioned functional modules may be the nucleotide sequences shown below or their functional equivalents or optimized variants. The nucleotide sequence of the 3xFLAG tag is shown in SEQ ID NO: 10. In some embodiments of the present invention, the nucleotide sequence of the SP6 promoter is shown in SEQ ID NO: 11. In some embodiments of the present invention, the nucleotide sequence of the EGFP gene is shown in SEQ ID NO: 12.

[0009] In some embodiments of the present invention, based on the above functional system, the present invention constructs three vectors: the first, the basic vector pWCF100-EGFP: it contains a complete dual-host replication system (pUC-ori and 256ori), a resistance screening system (ermB and its regulatory elements) and a target gene expression system (including a cloning site and a cbh terminator), and introduces an EGFP reporter gene and a 3xFLAG tag for expression verification. The second, the self-ligated vector pWCF100S-EGFP: it is obtained by self-ligation optimization design on the basis of the basic vector. In addition to retaining all the functional elements of the basic vector, the vector improves the replication efficiency in lactic acid bacteria through structural optimization and simplifies the cloning operation process. The third, the constitutive vector pWCF101S-EGFP: it is based on the self-ligated vector, integrates the P32 promoter sequence, and improves the expression regulation system. The vector contains a complete dual-host replication system, a resistance screening system, a target gene expression system, a labeling system (3xFLAG tag) and a verification system (EGFP gene), which realizes the constitutive and efficient expression of exogenous genes. Experimental verification shows that the vector system constructed by the present invention has the advantages of stable structure, high expression efficiency, simple operation and diverse functions. It can be stably inherited in the host bacteria and realize efficient secretory expression of foreign proteins. The cloning steps have been optimized and the construction efficiency has been significantly improved, which can meet different experimental needs.

[0010] In the second aspect of the present invention, a method for constructing the stable lactic acid bacteria secretory expression vector described in the first aspect is provided, the method comprising a process of constructing a basic vector, a process of constructing a self-ligating vector by self-ligation on the basis of the basic vector, and a process of further constructing a constitutive vector based on the self-ligating vector.

[0011] In an embodiment of the present invention, the method for constructing a basic vector includes: obtaining a DNA fragment of a replication system, wherein the replication system includes a lactic acid bacteria replicon sequence and an Escherichia coli replicon sequence; obtaining a DNA fragment of a resistance screening system, including a screening marker gene and its regulatory elements; obtaining a DNA fragment of a target gene expression system, including a secretion signal peptide sequence, a cloning site, and a terminator sequence; assembling the above DNA fragments into a single vector by a seamless cloning method to form the basic vector. Optionally, when constructing the basic vector, it also includes obtaining a DNA fragment of a marker system and / or a DNA fragment of a verification system, and introducing them into the basic vector by a seamless cloning method.

[0012] In an embodiment of the present invention, the method for constructing a self-ligating vector comprises: performing PCR amplification using the basic vector as a template to obtain a DNA fragment containing the full-length sequence of the basic vector; and self-ligating the DNA fragment containing the full-length sequence of the basic vector by a seamless cloning method to obtain the self-ligating vector.

[0013] In an embodiment of the present invention, the method for constructing a constitutive vector comprises: performing PCR amplification using the self-ligating vector as a template to obtain a first DNA fragment; obtaining a second DNA fragment containing a P32 promoter; and assembling the first DNA fragment and the second DNA fragment into a single vector by a seamless cloning method to form the constitutive vector.

[0014] In an embodiment of the present invention, the selection of each element in the replication system, resistance screening system and target gene expression system and their sequences may be as described in the first aspect above.

[0015] In one embodiment of the present invention, the present invention provides a method for constructing a stable lactic acid bacteria secretory expression vector. The method first constructs a basic vector, and obtains DNA fragments of a replication system, a resistance screening system, and a target gene expression system. Among them, the replication system includes a lactic acid bacteria replicon and an Escherichia coli replicon, which can be obtained by PCR amplification using pPG612 and pSIP403 plasmids as templates; the resistance screening system includes regulatory elements including the ermB gene and its promoter, leader peptide, and terminator; the target gene expression system includes a secretion signal peptide sequence, Nco I and Bam HI restriction site and cbh terminator sequence. The above DNA fragments are assembled into a single vector by seamless cloning to obtain the basic vector pWCF100-EGFP. In addition, when constructing the basic vector, the 3xFLAG tag of the labeling system and / or the verification system containing the SP6 promoter and the EGFP gene can be selectively introduced.

[0016] In one embodiment of the present invention, on the basis of obtaining the basic vector pWCF100-EGFP, the present invention further constructs a self-ligating vector. The specific method is to use the basic vector as a template for PCR amplification to obtain a DNA fragment containing the full-length sequence of the basic vector, and then self-ligate the DNA fragment by a seamless cloning method to obtain a self-ligating vector pWCF100S-EGFP. This step simplifies the cloning process and improves the construction efficiency.

[0017] In one embodiment of the present invention, in order to achieve constitutive expression of exogenous genes, the present invention also constructs a constitutive vector. The specific method is to use the self-ligating vector as a template to perform PCR amplification to obtain a first DNA fragment, and at the same time obtain a second DNA fragment containing a P32 promoter sequence, and assemble these two DNA fragments into a single vector by a seamless cloning method to form a constitutive vector pWCF101S-EGFP.

[0018] The seamless cloning technology used in the present invention avoids the limitations of traditional enzyme cutting and ligation methods and improves cloning efficiency. In actual operation, the PCR reaction uses a high-fidelity enzyme for amplification, and specific amplification products can be obtained after the reaction conditions are optimized. The cloned vectors are all verified by double enzyme cutting and PCR, and the correctness of the sequence is confirmed by sequencing. The experimental results show that the three vectors constructed by this method have good stability and functionality, and can meet different experimental requirements.

[0019] In the third aspect of the present invention, the application of the stable lactic acid bacteria secretory expression vector described in the first aspect in the construction of recombinant lactic acid bacteria is provided. The stable lactic acid bacteria secretory expression vector of the present invention can be effectively used to construct recombinant lactic acid bacteria. For example, in one embodiment of the present invention, the stable lactic acid bacteria secretory expression vector constructed by the present invention is transformed into plant lactobacillus NC8, and a recombinant strain with stable expression can be obtained through antibiotic screening. Specifically, the transformation process includes the steps of preparing lactic acid bacteria competent state, electrotransformation and strain screening.

[0020] The present invention verifies the expression in the recombinant lactic acid bacteria by Western blot experiment. The results show that the recombinant strain can stably express the target protein. For example, in one embodiment, the EGFP reporter protein fused with a Flag tag of about 31.8 kDa can be stably expressed, and the protein expression amount shows regular changes with the extension of the culture time. In the process of continuous subculture, the genetic stability of the recombinant strain is good, and the EGFP gene can still be stably maintained and the expression level can be maintained after 7 generations of continuous subculture.

[0021] In addition, the vector system of the present invention has good applicability in different types of lactic acid bacteria, and suitable host strains can be selected according to actual needs, providing an important tool for constructing various types of recombinant lactic acid bacteria. The recombinant lactic acid bacteria constructed by the vector of the present invention have the characteristics of good genetic stability and high expression efficiency, and can be applied to the fields of vaccine vector development, protein expression and functional research.

[0022] In the fourth aspect of the present invention, the application of the stable lactic acid bacteria secretory expression vector described in the first aspect above in expressing foreign genes is provided. The stable lactic acid bacteria secretory expression vector of the present invention can be effectively applied to the expression of foreign genes. For example, taking the EGFP reporter gene as an example, it is confirmed by Western blot experiments that the vector system can achieve stable expression of foreign genes in Escherichia coli Top10 and Lactobacillus plantarum NC8. For example, in Escherichia coli, the target protein can be expressed in the bacterium or secreted into the culture medium supernatant; in Lactobacillus plantarum NC8, the target protein can be detected from the beginning of culturing 4h, and the expression is continuously maintained at subsequent time points such as 6h and 8h, indicating that the system has the characteristics of rapid startup and continuous and stable expression, which fully reflects the advantages of the expression system driven by the P32 promoter in terms of timeliness and stability.

[0023] The vector system of the present invention has multiple advantages: first, through the optimized secretion signal peptide sequence, efficient secretory expression of foreign proteins can be achieved; second, the vector contains a 3xFLAG tag system, which is convenient for detecting and purifying the target protein; third, through the regulation of the P32 promoter, constitutive expression of foreign genes can be achieved. These characteristics enable the vector system to meet the needs of different types of foreign gene expression.

[0024] In the fifth aspect of the present invention, an engineered strain containing the stable lactic acid bacteria secretion expression vector described in the first aspect is provided. The engineered strain is preferably lactic acid bacteria, more preferably Lactobacillus plantarum. For example, in some embodiments of the present invention, the engineered strain constructed by the present invention includes Escherichia coli Top10 and Lactobacillus plantarum NC8 transformed with the stable lactic acid bacteria secretion expression vector. Among them, the Escherichia coli engineered strain containing pWCF100-EGFP, pWCF100S-EGFP or pWCF101S-EGFP can be used for vector construction and amplification; the Lactobacillus plantarum engineered strain containing pWCF101S-EGFP can be used for the expression and functional study of exogenous proteins.

[0025] The stability experiment verified that the constructed engineered strain had good genetic stability. After 7 generations of continuous subculture, PCR detection showed that the size of the target gene fragment remained unchanged, and Western blot results confirmed that the protein expression level was stable. In addition, the growth characteristics of the engineered strain were not significantly affected, indicating that the vector system had no obvious negative impact on the host strain.

[0026] The above invention content fully demonstrates the structural characteristics, construction method and application advantages of the stable lactic acid bacteria secretion expression vector provided by the present invention, and provides important technical support for the development of new microecological preparations and vaccine vectors.

[0027] Compared with the prior art, the beneficial effects or advantages of the present invention include: the present invention provides a stable lactic acid bacteria secretion expression vector and a construction method thereof. The vector system is realized through two-step optimization: first, the basic vector (pWCF100-EGFP) is transformed into a self-ligation vector (pWCF100S-EGFP) through self-ligation optimization to enhance the replication ability of lactic acid bacteria; then, the P32 promoter and secretion signal peptide are integrated to optimize it into a constitutive vector (pWCF101S-EGFP), realizing the characteristics of dual-host replication, strong promoter-driven expression and efficient secretion.

[0028] The present invention adopts seamless cloning technology and self-ligation strategy to construct vector, simplifies cloning process and improves construction efficiency. Experiments show that recombinant lactic acid bacteria can still stably maintain target gene expression after 7 generations of continuous passage, and Western blot detection shows that the expression of EGFP reporter protein fused with Flag tag is stable. In plant lactobacillus NC8, the expression of target protein can be detected in 4h and remains stable at subsequent time points, reflecting the timeliness and stability advantages of P32 promoter driven expression system.

[0029] The vector system of the present invention has dual replication ability (can replicate in Escherichia coli and lactic acid bacteria), combined with perfect screening markers and multiple detection methods (such as 3×FLAG tags and EGFP reporter genes), and can be widely used in vaccine vector development, protein expression and functional research and other fields. In summary, the present invention has significant application value in the field of recombinant lactic acid bacteria construction and exogenous gene expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein: Figure 1 The construction verification of the basic vector pWCF100-EGFP is shown. A shows the electrophoresis detection results of the PCR products of pPG612-EGFP and PSIP403; wherein, M: DL5,000 DNA Marker; DL10,000 DNA Marker; 1: pPG612-EGFP PCR product; 2: PSIP403 PCR product. B shows the electrophoresis results of the double enzyme digestion of pWCF100-EGFP, wherein, M: DL5,000 DNA Marker; DL10,000 DNA Marker; 1: pWCF100-EGFP double enzyme digestion product. C shows the electrophoresis results of the PCR product of pWCF100-EGFP; wherein, M: DL5,000 DNA Marker; 1: pWCF100-EGFP PCR product.

[0031] Figure 2The construction verification of the self-ligating vector pWCF100S-EGFP is shown. A shows the PCR electrophoresis results of pWCF100S-EGFP; M: DL5,000 DNA Marker; 1: pWCF100-EGFP PCR. B shows the electrophoresis results of double restriction enzyme digestion of pWCF100S-EGFP; M: DL5,000 DNA Marker; 1: pWCF100S-EGFP double restriction enzyme digestion product.

[0032] Figure 3 The construction verification of the constitutive vector pWCF101S-EGFP is shown. A shows the PCR electrophoresis results of pWCF100S, wherein M: DL5,000 DNA Marker; 1: pWCF100S PCR product. B shows the PCR electrophoresis results of pMG36e, wherein M: DL5,000 DNA Marker; 1: pMG36e PCR product. C shows the electrophoresis results of pWCF101S-EGFP double restriction enzyme digestion. wherein M: DL5,000 DNA Marker; 1: pWCF101S-EGFP double restriction enzyme digestion. D shows the electrophoresis results of pWCF101S-EGFP PCR. wherein M: DL5,000 DNA Marker; 1: pWCF101S-EGFP PCR product.

[0033] Figure 4 The construction verification of the empty vector pWCF101S-EE is shown. A shows the PCR electrophoresis results of pWCF101S-V1; wherein, M: DL5,000 DNA Marker; 1: pWCF101S-V1 PCR product. B shows the double enzyme digestion electrophoresis results of pWCF101S-EE; wherein, M: DL5,000 DNA Marker; 1: pWCF101S-EE double enzyme digestion product. C shows the PCR electrophoresis results of pWCF101S-EE. wherein, M: DL5,000 DNA Marker; 1: pWCF101S-EE PCR product.

[0034] Figure 5Western blot detection bands are shown. A shows the Western blot identification results of Top10 supernatant expression. M: Marker; 1: Top10-pWCF101S-EE; 2: Top10-pWCF101S-EGFP. B shows the Western blot identification results of Top10 precipitate expression; M: Marker; 1: Top10-pWCF101S-EGFP 2: Top10-pWCF101S-EE. C shows the Western blot identification results of Top10 precipitate expression; M: Marker; 1: Top10-pWCF101S-EGFP 2: Top10-pWCF101S-EE. D shows the results of Western blot identification of NC8 supernatant expression (Flag antibody); wherein, M: Marker; 1: NC8-pWCF101S-EE; 2: NC8-pWCF101S-EGFP 4h; 3: NC8-pWCF101S-EGFP 6h; 4: NC8-pWCF101S-EGFP 8h. E shows the results of Western blot identification of NC8 supernatant expression (EGFP antibody); wherein, M: Marker; 1: NC8-pWCF101S-EE; 2: NC8-pWCF101S-EGFP 4h; 3: NC8-pWCF101S-EGFP 6h; 4: NC8-pWCF101S-EGFP 8h.

[0035] Figure 6Stability verification bands are shown. A shows the results of NC8 pWCF101S-EGFP PCR electrophoresis detection; M: Marker; 1: 1st generation NC8 pWCF101S-EGFP; 2: 2nd generation NC8 pWCF101S-EGFP; 3: 3rd generation NC8 pWCF101S-EGFP; 4: 4th generation NC8 pWCF101S-EGFP; 5: 5th generation NC8 pWCF101S-EGFP; 6: 6th generation NC8 pWCF101S-EGFP; 7: 7th generation NC8 pWCF101S-EGFP. B shows the results of Western blot identification of NC8 supernatant expression; wherein, M: Marker; 1: NC8-pWCF101S-EE; 2: 1st generation NC8 pWCF101S-EGFP; 3: 2nd generation NC8 pWCF101S-EGFP; 4: 3rd generation NC8 pWCF101S-EGFP; 5: 4th generation NC8 pWCF101S-EGFP; 6: 5th generation NC8 pWCF101S-EGFP; 7: 6th generation NC8 pWCF101S-EGFP; 8: 7th generation NC8 pWCF101S-EGFP. DETAILED DESCRIPTION

[0036] The present invention is described in conjunction with specific examples. It should be understood that the examples are only used to illustrate and not to limit the present invention. Experimental methods without specifying specific conditions are carried out according to conventional conditions or manufacturer recommendations. The professional terms used in the present invention have conventional meanings in the art, and the reagents and raw materials used can be obtained by conventional means. In addition, any similar or equivalent methods and materials can be used in the present invention, and the embodiments are only examples.

[0037] Example Construction and expression verification of recombinant lactic acid bacteria expressing ETEC F4+ antibody 1.1 Materials 1.1.1 Strains and plasmid materials: pPG612-EGFP, pSIP403 and pMG36e were preserved and provided by the Engineering Research Center for Major Animal Disease Microecological Vaccines (Drugs) of Jilin Agricultural University, Ministry of Education.

[0038] 1.1.2 Enzymes and reagents: restriction endonucleases Nco I and BmI. PrimeSTAR Max Premix (2×), DNAMarker (DL2000, DL5000, DL10000), and 10xLoading Buffer were purchased from Yubao Bioengineering (Dalian) Co., Ltd. (Takara); seamless cloning reagent ClonExpress II One Step Cloning Kit was purchased from Vazyme; BCA protein concentration determination kit (enhanced) was purchased from Bio-Tech Biotechnology Co., Ltd.; Plasmid Mini Kit I was purchased from OMEGA; prestained protein labeling, ECL chemiluminescence kit, multiple recombinant HRP-labeled goat anti-mouse secondary antibody, and anti-DYKDDDDK tag monoclonal antibody were purchased from Wuhan Proteintech Co., Ltd. (Proteintech); other reagents required for the experiment were domestic analytical grade products or imported.

[0039] 1.1.3 Main instruments and equipment for the experiment: pipettes and gradient PCR instrument were purchased from Eppendorf, Germany; fully automatic autoclave GR85DA was purchased from Sigma, Germany; analytical balance was selected from LEAPING brand; Mini-PROTEAN Tetra small vertical electrophoresis system was provided by DiTu (Shanghai) Biotechnology Co., Ltd. In addition, the Gene Pulser XcellTM System electroporator and Universal Hood Ⅱ gel imaging analysis system used in the experiment were both from BIO-RAD, USA, and the AI600 chemiluminescence imaging system was produced by GE, USA.

[0040] 1.1.4 Test reagents and preparation 1.1.4.1 Preparation of culture medium: (1) LB liquid culture medium: Weigh 10.0 g tryptone, 10.0 g yeast extract and 5.0 g sodium chloride, add 800 L double distilled water (ddH2O) to dissolve, dilute to 1 L after dissolution, and stir evenly. Dispense an appropriate amount into test tubes or conical flasks, seal the bottle mouth with sealing film, place in an autoclave, sterilize at 121℃ for 15 min, wait for it to cool, and store at room temperature. (2) LB solid culture medium: Add agar powder to liquid LB, 1.5 g / 100 mL each, so that the final concentration is 1.5% (M / V), dispense an appropriate amount into conical flasks, seal the bottle mouth with sealing film, place in an autoclave, sterilize at 121℃ for 15 min, wait for it to cool, take out, and store at room temperature. (3) MRS liquid culture medium: Each liter of culture medium contains 10.0 g of peptone, 8.0 g of beef extract powder, 4.0 g of yeast extract, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, and 1 mL of Tween 80. Dissolve the above components in 0.7 L of deionized water, and after fully dissolving, dilute to 1 L and stir evenly. Dispense the prepared culture medium into test tubes or conical flasks in appropriate amounts and seal the flasks with sealing film. Then place it in an autoclave and sterilize it at 118°C for 15 minutes. After the culture medium cools, store it at room temperature. (4) MRS solid medium: Add agar powder to liquid MRS at a concentration of 1.8 g / 100 mL to a final concentration of 1.8% (M / V). Dispense appropriate amounts into conical bottles, seal the bottles with sealing film, place in an autoclave, sterilize at 118°C for 15 min, wait for it to cool, then remove and store at room temperature.

[0041] 1.1.4.2 Preparation of nucleic acid electrophoresis reagents: (1) Preparation of TAE (50×) electrophoresis buffer: Weigh 242 g Tris, add 57.1 mL acetic acid and 100 mL 0.5 mol / L EDTA solution (pH 8.0), and dissolve with 700 L double distilled water. After the reagent is completely dissolved, dilute to 1 L with double distilled water. This buffer can be stored at room temperature. (2) Preparation of agarose gel (1%): Take 100 mL 1×TAE buffer and add 1 g agarose. Stir under heating conditions to completely dissolve the agarose. When the solution cools to about 60°C, add 10 μL of nucleic acid dye and mix well. Then pour the solution into a gel tray with an electrophoresis comb pre-inserted, and solidify at room temperature for about 30 minutes before use.

[0042] 1.1.4.3 Preparation of Western blot reagents: (1) 5×SDS-PAGE electrophoresis buffer: Weigh 15.1 g Tris and 94.0 g glycine, dissolve in 700 mL double distilled water, and then add 50 mL 10% SDS solution. After complete dissolution, dilute to 1 L with double distilled water and adjust the pH to 8.3. This buffer can be stored at room temperature and needs to be diluted when used. (2) Transfer buffer: Dissolve 5.8 g Tris, 2.9 g glycine and 0.37 g SDS in 600 mL double distilled water, dilute to 800 mL after dissolution, and finally add 200 mL methanol. This solution can be stored at room temperature. (3) 10×TBS solution: Weigh 236 g sodium chloride (NaCl) and 72.6 g Tris, dissolve in 2 L double distilled water, and then dilute to 3 L. Use concentrated hydrochloric acid to adjust the pH to 7.6. (4) TBST solution: Take 100 mL 10×TBS and 1 mL Tween-20, dilute to 1 L with double distilled water and mix well. This solution needs to be stored in a refrigerator at 4°C. (5) Blocking buffer: Dissolve 5 g skim milk powder in 100 mL TBST. After dissolution, filter and sterilize using a 0.22 μm filter membrane, divide into sterile 1.5 mL EP tubes, and store at -20°C. It is recommended to prepare this solution before use.

[0043] 1.1.4.4 Preparation of competent reagent (SOB reagent): Weigh 2 g tryptone, 0.5 g yeast extract, 0.05 g sodium chloride, 0.0186 g potassium chloride, 0.203 g magnesium chloride hexahydrate and 0.49 g magnesium sulfate heptahydrate, and dissolve them in 80 μL double distilled water. After complete dissolution, dilute to 100 mL with double distilled water, adjust the pH to 7.5 with sodium hydroxide, and then sterilize at 121°C for 15 minutes.

[0044] 1.1.4.5 Other related reagents for the experiment: (1) Preparation of 80% glycerol: Mix 80 mL of glycerol with 20 mL of double distilled water and sterilize at 121°C for 15 minutes. After the solution cools, store it at 4°C. (2) Preparation of storage concentration of erythromycin: Dissolve 2 g of erythromycin in 100 mL of anhydrous ethanol. (2) Preparation of lysozyme: Weigh 1 g of lysozyme and dissolve it in 50 mL of double distilled water.

[0045] 1.1.5 The functional elements required for constructing a stable lactic acid bacteria secretory expression vector in the following example are as follows: Replication system: including Escherichia coli replicon sequence (pUC-ori) and lactic acid bacteria replicon sequence (256ori). Among them, the pUC-ori sequence is shown in SEQ ID NO: 3, which can be obtained by PCR amplification using pPG612 plasmid as a template; the 256ori sequence is shown in SEQ ID NO: 2, which can be obtained by PCR amplification using pSIP403 plasmid as a template. Resistance screening system: including ermB gene and its regulatory elements. The ermB gene sequence is shown in SEQ ID NO: 4, the ermB promoter sequence is shown in SEQ ID NO: 5, the ermB leader peptide sequence is shown in SEQ ID NO: 6, and the ermB terminator sequence is shown in SEQ ID NO: 7. These sequences can be obtained by PCR amplification using pPG612 plasmid as a template. Target gene expression system: including secretion signal peptide sequence and cbh terminator sequence. Among them, the secretion signal peptide sequence is shown in SEQ ID NO: 1, and the cbh terminator sequence is shown in SEQ ID NO: 8. These sequences can be obtained, for example, by PCR amplification using pSIP403 plasmid as a template. P32 promoter system: The P32 promoter sequence is shown in SEQ ID NO: 9, which can be obtained, for example, by PCR amplification using pMG36e plasmid as a template. Labeling and verification system: includes 3xFLAG tag, SP6 promoter and EGFP gene. Among them, the 3xFLAG tag sequence is shown in SEQ ID NO: 10, the SP6 promoter sequence is shown in SEQID NO: 11, and the EGFP gene sequence is shown in SEQ ID NO: 12. These functional sequences can be obtained, for example, by PCR amplification using pPG612-EGFP plasmid as a template. The above-mentioned functional elements can be orderly assembled and constructed into different types of vectors through reasonably designed PCR primers and seamless cloning technology. The PCR primer sequences and specific construction methods are described in Section 1.2 below.

[0046] 1.2 Methods 1.2.1 Primer sequences: In order to obtain the target gene, the following target gene cloning primers were designed, including: 7-612Forward (SEQ ID NO: 13), 7-612Reverse (SEQ ID NO: 14), 7-403Forward (SEQ ID NO: 15), 7-403Reverse (SEQ ID NO: 16), pWCF100chE2Forward (SEQ ID NO: 17), pWCF100chE2Reverse (SEQ ID NO: 18), pWCF101SelfcycleForward (SEQ ID NO: 19), pWCF101SelfcycleReverse (SEQ ID NO: 20), V-Forward (SEQ ID NO: 21), V-Reverse (SEQ ID NO: 22), P32-Forward (SEQ ID NO: 23), P32-Reverse (SEQ ID NO: 24), WYJ-101sys1Forward (SEQ ID NO: 25), WYJ-101sys1 Reverse (SEQ ID NO: 26), WYJ-V2checkForward (SEQ ID NO: 27), WYJ-V2check Reverse (SEQ ID NO: 28).

[0047] 1.2.2 Preparation of competent E. coli Top10: a. Inoculate the bacteria into a 5mL LB test tube, culture for a period of time, use a bacterial loop to streak LB, and culture overnight; b. Pick a single colony into a 5mL LB test tube and culture overnight; c. Take 500μL from the 5mL test tube of the previous day and add it to 100mL SOB medium, place it in a shaker at 37℃ 180rpm~220rpm for 2-3h until the OD 600=0.35-0.4, collect the bacteria, and pre-cool the centrifuge at 4℃; d. Divide 100mL of bacterial solution into 2 50mL centrifuge tubes and place on ice for 10min; e. Centrifuge at 4℃, 2200g for 10min; f. Discard the supernatant and completely aspirate the liquid with a 200μL pipette; g. Add 20mL of washing buffer to each 50mL centrifuge tube, resuspend the precipitate, and place on ice for 10min; h. Centrifuge at 4℃, 2000g for 10min; i. Discard the supernatant and completely aspirate the liquid with a 200μL pipette; j. Add 2mL of FSB to each 50mL centrifuge tube and gently suspend the precipitate; k. Add 70μL of DMSO to each 50mL centrifuge tube without waiting, gently rotate to mix, do not blow or aspirate with a pipette, and place on ice for 15min; l. Repeat the previous step completely; m. Divide into 1.5EP tubes, 100ul per tube. Keep the EP tubes upright on ice, quick-freeze the bottom bacteria with liquid nitrogen, and keep them in liquid nitrogen until stored in a -80℃ refrigerator.

[0048] 1.2.3 Cloning and verification of recombinant plasmid pWCF100-EGFP 1.2.3.1 Obtaining the DNA fragment required for cloning pWCF100-EGFP: The PCR amplification reaction system for pPG612 and pSIP403 is the same, with a total volume of 50 μL. The specific components are as follows: 0.5 μL of plasmid DNA template, 1 μL of forward primer, 1 μL of reverse primer, 25 μL of 2× PrimeSTAR® Mix, and 22.5 μL of double distilled water. The primers used for the two plasmids are 7-612 and 7-403 series primers, respectively. The PCR reaction conditions are as follows: first, pre-denaturation at 98℃ for 10 seconds, enter 30 cycles: denaturation at 98℃ for 10 seconds, annealing at 57℃ for 5 seconds, extension at 72℃ for 15 seconds, and final extension at 72℃ for 1 minute after the cycle ends, and finally keep at 4℃ constant temperature (∞). Detection method of PCR product: Take 10 μL of PCR product and mix it with 1 μL of 10× Loading Buffer, and perform electrophoresis on 1% agarose gel. The electrophoresis conditions were 120 V for 25 minutes, and the PCR product was verified by observing the band size. Take 10 μL of PCR product and add 1 μL of 10x Loading Buffer, and perform nucleic acid electrophoresis on 1% agarose gel at 120 V for 25 minutes to verify the band size of the plasmid PCR product.

[0049] 1.2.3.2 Cloning of recombinant plasmid pWCF100-EGFP: Use the PCR products of pPG612 and pSIP403 as templates to clone the pWCF100-EGFP plasmid. IN-Fusion cloning system (ligation reaction system for pWCF100-EGFP): Total volume 20 μL, take 1 μL pPG612 PCR product and 2 μL pSIP403 PCR product, add 4 μL 5×CE II Buffer and 2 μL Exnase II enzyme, and finally add double distilled water to 20 μL. Cloning and acquisition steps of ligation product: Transform the recombinant plasmid pWCF100-EGFP obtained by ligation reaction into Escherichia coli Top10 competent cells, and spread the transformation solution on LB solid medium containing 200 μg / mL erythromycin. Pick a single colony and inoculate it into LB liquid medium containing the same concentration of erythromycin for expansion culture. The target recombinant plasmid pWCF100-EGFP was obtained by extracting the plasmid according to the instructions of the OMEGA Plasmid Mini Kit I.

[0050] 1.2.3.3 Identification of recombinant plasmid pWCF100-EGFP: The correctness of pWCF100-EGFP plasmid was identified by restriction digestion and PCR. Nco I and Bm I double-enzyme digestion of pWCF100-EGFP plasmid. First, prepare 10 μL double-enzyme digestion reaction system, including 6 μL pWCF100-EGFP plasmid, 0.5 μL each Nco I and Bam HI restriction endonuclease, 1μL 10× Buffer, and finally 2μL double distilled water. The reaction system was placed in a 37℃ water bath for 2h. After the digestion, 9μL of the digestion product was mixed with 1μL 10× Loading Buffer, and electrophoresed on a 1% agarose gel at 120V for 30min. The correctness of the recombinant plasmid construction was verified by observing the DNA band size.

[0051] The pWCF100-EGFP plasmid was amplified by PCR. A 10 μL PCR reaction system was prepared, including 0.5 μL pWCF100-EGFP plasmid template, 0.5 μL each of the forward primer pWCF100chE2 Forward and the reverse primer pWCF100chE2 Reverse, 5 μL 2× PrimeSTAR® Mix, and finally 3.5 μL double distilled water. PrimeSTAR® Mix already contains the necessary PCR components such as DNA polymerase, reaction buffer and dNTPs, and no additional addition is required.

[0052] PCR was used to identify whether the pWCF100-EGFP plasmid contained the EGFP reporter gene. The PCR reaction program was: 98℃ pre-denaturation for 10s; followed by 30 cycles, including 98℃ denaturation for 10s, 58℃ annealing for 5s, and 72℃ extension for 27s; finally 72℃ extension for 1min, and finally kept at 4℃ constant temperature (∞). Take 9μL of PCR product and add 1μL of 10× Loading Buffer, perform electrophoresis at 120V for 25min on 1% agarose gel to verify the size of the PCR product band.

[0053] 1.2.4 Cloning and verification of recombinant plasmid pWCF100S-EGFP 1.2.4.1 The recombinant plasmid pWCF100-EGFP was subjected to PCR using primers pWCF101SelfcycleF and primer pWCF101SelfcycleF. pWCF100-EGFP plasmid reaction system: the total volume is 50μL, and the specific components are as follows: pWCF100-EGFP plasmid 0.5μL, pWCF101Selfcycle Forward primer 1μL, pWCF101SelfcycleReverse primer 1μL, 2×PrimeSTAR® Mix 25μL, ddH2O 22.5μL. The PCR reaction conditions are as follows: first, pre-denaturation at 98°C for 10s, followed by 30 cycles, each cycle including denaturation at 98°C for 10s, annealing at 57°C for 5s, and extension at 72°C for 27s; after the cycle, extension at 72°C for 1min; finally, storage at 4°C until taken out.

[0054] 1.2.4.2 Seamlessly clone the PCR product from the previous step. The pWCF100S-EGFP ligation reaction was performed in a total volume of 20 μL. The specific components were as follows: 2 μL of pWCF100 PCR product, 4 μL of 5 × CE II Buffer, 2 μL of Exnase II, and 12 μL of ddH2O was added to make up to the final volume.

[0055] 1.2.4.3 Enzyme digestion verification of recombinant plasmid pWCF100S-EGFP: After transforming the connected recombinant plasmid pWCF100S-EGFP into Top10, pick a single colony on a plate and expand the culture. Then extract the plasmid according to the instructions of the plasmid small-scale extraction kit (universal type). After obtaining the recombinant plasmid pWCF100S-EGFP, perform enzyme digestion verification. The total volume of the enzyme digestion verification system is 10μL, and the specific components are as follows: pWCF100S-EGFP 6μL, Nco I and BamHI restriction enzyme 0.5 μL each, 10× Buffer 1 μL, ddH2O 2 μL. After the reaction system was digested at 37°C for 2 hours, 1 μL 10× Loading Buffer was added, and then nucleic acid electrophoresis was performed on a 1% agarose gel at 120V for 30 minutes to verify whether the band size of the recombinant plasmid pWCF100S-EGFP was in line with expectations.

[0056] 1.2.5 Cloning and verification of recombinant plasmid pWCF101S-EGFP pWCF100S PCR reaction system: total volume 50μL, components: pWCF100S plasmid 0.5μL, V-Forward primer 1μL, V-Reverse primer 1μL, 2× PrimeSTAR® Mix 25μL, ddH2O 22.5μL. PCR reaction conditions: first pre-denaturation at 98°C for 10s, followed by 30 cycles, each cycle including denaturation at 98°C for 10s, annealing at 57°C for 5s, and extension at 72°C for 22s; after the cycle, extend at 72°C for 1min; finally, store at 4°C until taken out. Take 10μL of PCR product and add 1μL 10× Loading Buffer, and perform electrophoresis on 1% agarose gel at 120V for 25min to verify the size of the pWCF100S plasmid PCR product band.

[0057] pMG36e PCR reaction system: total volume 50μL, components as follows: pMG36e plasmid 0.5μL, P32-Forward primer 1μL, P32-Reverse primer 1μL, 2× PrimeSTAR® Mix 25μL, ddH2O 22.5μL. PCR reaction conditions are as follows: first, pre-denaturation at 98°C for 10s, followed by 30 cycles, each cycle including denaturation at 98°C for 10s, annealing at 57°C for 5s, and extension at 72°C for 20s; after the cycle, extension at 72°C for 1min; finally, storage at 4°C until removal. Take 10μL of PCR product and add 1μL 10× Loading Buffer, and perform electrophoresis on 1% agarose gel at 120V for 25min to verify the size of the pMG36e plasmid PCR product band.

[0058] The total volume of the seamless cloning connection (pWCF101S-EGFP connection reaction) reaction system is 20μL, and the specific components are as follows: pWCF100S PCR product 1μL, pMG36e PCR product 2μL, 5 × CE II Buffer 4μL, Exnase II 2μL, ddH2O 11μL. After completing the connection reaction, the recombinant plasmid pWCF101S-EGFP was transformed into Top10 competent cells, and a single colony was picked for expansion culture after plating. Extract the plasmid according to the instructions of the plasmid small-scale extraction kit (universal type) to obtain the recombinant plasmid pWCF101S-EGFP, and then perform enzyme cutting verification. The total volume of the enzyme cutting verification system is 10μL, and the components are as follows: pWCF101S-EGFP 6μL, Nco I and Bam HI restriction enzyme 0.5 μL each, 10× Buffer 1 μL, ddH2O 2 μL. After the reaction system was digested at 37°C for 2 hours, 1 μL 10× Loading Buffer was added, and then nucleic acid electrophoresis was performed on a 1% agarose gel at 120V for 30 minutes to verify whether the band size of the recombinant plasmid pWCF101S-EGFP was in line with expectations. Finally, the plasmid was sent to Sangon for sequencing, and the sequencing results showed that the sequence was 100% correct without any mutations.

[0059] 1.2.6 Preparation of competent culture of Lactobacillus plantarum NC8: (1) Precool the electroporation buffer and washing buffer in a 4℃ refrigerator, and precool the 1.5mL EP tube in a -80℃ refrigerator. (2) Take the frozen NC8 bacteria and place them in 5mL MRS liquid culture medium, and anaerobically activate and resuscitate them at 37℃ for 12h. (3) Use a bacterial loop to dip the bacterial liquid and streak it on the MRS solid culture medium, and anaerobically culture it at 37℃ for 16-18h. (4) Pick a single NC8 colony and inoculate it in 5mL MRS liquid culture medium, add 100μL 2%Gly and anaerobically culture it at 37℃ for 7-8h. (5) Take 2mL of bacterial liquid and inoculate it in 100mL MRS liquid culture medium, add 2mL 2%Gly and anaerobically culture it at 37℃ until OD 600 =0.2-0.3. (6) Transfer the bacterial suspension to two 50mL centrifuge tubes, place on ice for 10min, centrifuge at 4℃ 5000rpm for 10min, and discard the supernatant. (7) Resuspend the precipitate in 10mL pre-cooled washing buffer in each tube, centrifuge at 4℃ 5000rpm for 10min, and discard the supernatant. (8) Repeat this process, resuspend the precipitate in pre-cooled washing buffer, centrifuge at 4℃ 5000rpm for 10min, and discard the supernatant. (9) Resuspend the bacterial cells in 1mL electroporation buffer in each tube, place on ice for 10min, and dispense 100μL / tube into 1.5mL EP tubes. Quickly freeze in liquid nitrogen and store in a -80℃ refrigerator.

[0060] 1.2.7 Transformation and storage of recombinant plasmid: (1) Soak the 2mm electroporation cup in 75% alcohol overnight. (2) Dry completely under UV light and precool on ice. (3) Take out the NC8 competent bacteria from the -80℃ refrigerator and place it on ice. When the competent bacteria are in an ice-water mixture state, add 1μL of plasmid, mix gently, transfer to the electroporation cup, and place on ice for 10 minutes. (4) Set the electroporation system conditions to output voltage 2000V, capacitance 25μF, sample resistance 400Ω, and the distance between the electrodes of the electroporation cup is 2mm. After the electroporation, let it stand at room temperature for 3 minutes. (5) Add 600μL of 30℃ preheated MRS liquid culture medium and 150μL of 0.5mol / L sucrose, mix well, transfer to a 1.5mL centrifuge tube, and culture in a 30℃ water bath for 3 hours to recover. (6) Centrifuge at 4000rpm for 5min, discard 600μL of liquid, mix the bacterial precipitate, and plate on MRS solid medium with 10μg / mL erythromycin. Incubate anaerobically at 37℃. Place upright for 1h and then invert for 17h. (7) Pick a single colony and incubate anaerobically at 37℃ in 5mL MRS liquid medium with 10μg / mL erythromycin for 12h. (8) Take 800μL of bacterial solution and add 200μL 80% glycerol and store in a -80℃ refrigerator. (9) Extract the plasmid from the remaining bacterial solution. After suspending the bacterial precipitate, add 180μL 50μg / μL lysozyme and incubate at 37℃ on a shaker for 2h. The remaining steps are performed according to the instruction manual of the Plasmid Mini-Extraction Kit (Universal Type). The plasmid is stored in a -20℃ refrigerator.

[0061] 1.2.8 PCR verification of recombinant plasmid pWCF101S-EGFP in NC8: After transforming the recombinant plasmid pWCF101S-EGFP proposed in Top10 in the previous step into NC8, spread it on MRS solid medium containing erythromycin with a coating rod, culture it overnight, pick a single colony, expand the culture, and then extract the plasmid according to the instructions of the plasmid small-scale extraction kit (universal type). After obtaining the recombinant plasmid pWCF101S-EGFP, perform PCR verification. pWCF101S-EGFP PCR reaction system: The total volume of the PCR reaction system is 10μL, and the components are: pWCF101S-EGFP plasmid 0.5μL, pWCF100chE2 Forward primer 0.5μL, pWCF100chE2 Reverse primer 0.5μL, 2×PrimeSTAR® Mix 5μL, ddH2O 3.5μL. The PCR reaction conditions were as follows: first, pre-denaturation at 98°C for 10s, followed by 30 cycles, each cycle including denaturation at 98°C for 10s, annealing at 57°C for 5s, and extension at 72°C for 5s; after the cycle, extension at 72°C for 1min; and finally, storage at 4°C until removal. The PCR product was added with 1μL 10× Loading Buffer and electrophoresed on a 1% agarose gel at 120V for 25min to identify whether the gene plasmid pWCF101S-EGFP PCR product band size was correct.

[0062] 1.2.9 Preparation and verification of empty vector strains 1.2.9.1 Cloning and verification of Top10-pWCF101S-EE empty vector: The total volume of the pWCF100S-V1 PCR reaction system was 10 μL, and the components included 0.5 μL of pWCF101S-V1 plasmid, 0.5 μL of WYJ-101sys1 Forward primer, 0.5 μL of WYJ-101sys1 Reverse primer, 5 μL of 2× PrimeSTAR® Mix, and 3.5 μL of ddH2O. The PCR reaction conditions were as follows: first, pre-denaturation at 98°C for 10 s, followed by 30 cycles, each cycle including denaturation at 98°C for 10 s, annealing at 59°C for 5 s, and extension at 72°C for 20 s; after the cycle, extension at 72°C for 1 min; and finally, storage at 4°C until removal. Add 1 μL 10× Loading Buffer to the PCR product and perform electrophoresis on a 1% agarose gel at 120V for 25 minutes to verify whether the band size of the recombinant plasmid pWCF101S-EGFP PCR product is correct. After transforming the connected recombinant plasmid pWCF101S-EE into Top10, pick a single colony on the plate and expand the culture. Then extract the plasmid according to the instructions of the plasmid small-scale extraction kit (universal type). After obtaining the recombinant plasmid pWCF101S-EE, perform enzyme digestion verification. The total volume of the enzyme digestion verification system is 10 μL, and the specific components are as follows: pWCF101S-EE 6 μL, Nco I and Bam HI restriction enzyme 0.5 μL each, 10×Buffer 1 μL, ddH2O 2 μL. After the reaction system was digested at 37°C for 2 hours, 1 μL 10×Loading Buffer was added, and then nucleic acid electrophoresis was performed on a 1% agarose gel at 120V for 30 minutes to confirm that there was no target gene on the vector. Finally, the plasmid was sent to Sangon for sequencing, and the sequencing results showed that the sequence was correct and there was no mutation.

[0063] 1.2.9.2 Transformation and verification of NC8-pWCF101S-EE empty vector strain: The verified pWCF101S-EE empty vector plasmid was transformed into Lactobacillus plantarum NC8, and the transformation steps were the same as 1.2.7. Apply to MRS solid medium containing erythromycin, culture overnight, pick a single colony, expand the culture, and then extract the plasmid according to the instructions of the plasmid small-scale extraction kit (universal type), and perform PCR cloning of the tag gene fragment without any target gene to verify whether the transformation is successful. The total volume of the PCR reaction system is 10μL, and the specific components are as follows: pWCF101S-EE plasmid 0.5μL, WYJ-V2check Forward primer 0.5μL, WYJ-V2check Reverse primer 0.5μL, 2×PrimeSTAR® Mix 5μL, ddH2O 3.5μL. The PCR reaction conditions were as follows: first, pre-denaturation at 98°C for 10s, followed by 30 cycles, each cycle including denaturation at 98°C for 10s, annealing at 59°C for 5s, and extension at 72°C for 5s; after the cycle, extension at 72°C for 1min; and finally storage at 4°C until removal. The PCR product was added with 1μL 10× Loading Buffer and electrophoresed on a 1% agarose gel at 120V for 25min to verify whether the PCR product band size was correct.

[0064] 1.2.10 Verification of protein expression of recombinant plasmid pWCF101S-EGFP in Top10 1.2.10.1 Sample processing: (1) Activate Top10 stored in a -80℃ refrigerator, transfer to 5mL LB liquid culture medium and culture overnight at 37℃ in a shaking incubator; (2) On the next day, extract 500μL from 5mL LB liquid culture medium into 50mL culture medium and culture at 30℃; (3) Place the cultured Top10 on ice, cool for 10min, and separate the culture supernatant and precipitate by centrifugation at 2500g for 10min at 4℃; Pass the supernatant through a 0.22nm filter membrane, and concentrate it using an ultrafiltration column after filtration; (4) Add 5mL PBS solution to the precipitate and suspend it, centrifuge it at 2500g for 10min at 4℃, and repeat twice; (5) Take 1mL of bacterial culture, centrifuge it at 2500g for 10min, and suspend the precipitate with 1mL lysis buffer; (6) Transfer it to 2mL of Lysing Matrix Tube lysis tube containing 0.05-0.1mm glass beads; (7) pass through a grinding machine, 7000Hz, 20s, repeated 5 times, and placed on ice for 5min between each time; (8) centrifuge at 10000g for 3min at 4℃; (9) take 200μL of the ground bacterial supernatant and concentrated culture medium supernatant, add 50μL 5×SDS PAGE Buffer and mix well; (10) clamp the centrifuge tube with an explosion-proof clamp and boil in a 100℃ boiling water bath for 10min, then centrifuge at 12000rpm for 2min, use the supernatant for sample loading, and the sample can be stored at -20℃.

[0065] 1.2.10.2 BCA protein quantification: Calculate the concentration of the sample to be tested by using the BCA protein concentration determination kit (enhanced).

[0066] 1.2.10.3 Western blot verification of target protein expression: FLAG / EGFP was used to verify whether the new functional lactic acid bacteria expressed the target protein. (1) Sample addition: Add 10 μL of the processed sample and marker to the gel well; (2) Electrophoresis: Connect the power supply, voltage 120V, electrophoresis until the bromophenol blue line reaches the bottom of the electrophoresis tank and stop; (3) Gel treatment: Use a gel remover to gently pry open the gel glass plate, take out the gel, use a gel stripper to trim the gel, remove the concentrated gel, and soak it in the transfer solution; (4) Transfer: Immerse the transfer splint in the transfer solution, black side down, and place the transfer sponge pad gel and NC membrane transfer sponge pad in turn, use a transfer roller to drive out the bubbles between the gel and the NC membrane, clamp the transfer splint, place it in the transfer tank with the transfer solution, put in an ice box and a small rotor, place the transfer tank on the stirrer, and transfer at a constant current of 400 mA for 0.5 h. (5) Blocking: Take out the NC membrane and place it in the antibody incubation box, add 5% skim milk powder and block it on a shaker at room temperature for 1 hour. Add TBST to wash the membrane at room temperature for 10 minutes, repeat 3 times, and pour it out and replace it with a new one each time; (6) Incubate with primary antibody: Add mouse FLAG / EGFP primary antibody and incubate at 4℃ for 13 hours. Recover the antibody and store it at -20℃, add TBST to wash the membrane at room temperature for 10 minutes, repeat 3 times, pour it out and replace it with a new one each time; (7) Incubate with primary antibody: Add anti-mouse IgG HRP secondary antibody and incubate at room temperature for 1 hour. Recover the antibody and store it at -20℃, add TBST to wash the membrane at room temperature for 10 minutes, repeat 3 times, pour it out and replace it with a new one each time; (8) Place the membrane in the imaging system, add developer, and expose it.

[0067] 1.2.11 Verification of protein expression of recombinant plasmid pWCF101S-EGFP in NC8 1.2.11.1 Sample processing: (1) Activate NC8 stored in a -80℃ refrigerator, transfer to 5mL MRS liquid culture medium and culture overnight at 37℃ anaerobic workstation; (2) On the next day, extract 500μL from 5mL LB liquid culture medium into 50mL culture medium and culture at 30℃ for 4h, 6h, and 8h; (3) Place the cultured NC8 on ice, cool for 10min, centrifuge at 4℃ 2500g for 10min, and take the supernatant; (4) Pass the supernatant through a 0.22nm filter membrane, and concentrate it using an ultrafiltration column after filtration; (5) Take 200μL of the concentrate and add 50μL 5×SDS PAGE Buffer to mix; (6) Clamp the centrifuge tube with an explosion-proof clamp and boil it in a 100℃ boiling water bath for 10min, then centrifuge at 12000rpm for 1min, use the supernatant to load the sample, and the sample can be stored at -20℃.

[0068] 1.2.11.2 BCA protein quantification: Same as 1.2.10.2. Western blot verification of target protein expression: Same as 1.2.10.3.

[0069] 1.3 Results 1.3.1 Vector construction 1.3.1.1 Cloning and verification of the recombinant expression plasmid pWCF100 in Top10: PCR was performed using pPG612 and pSIP403 as templates to obtain linear DNA of 2655 bp and 2521 bp, respectively. Figure 1 In A, the pPG612-EGFP PCR product band (containing the E. coli replicon and resistance screening system) can be seen at 2521 bp in channel 1, and the PSIP403 PCR product band (containing the lactic acid bacteria replicon and target gene expression system) can be seen at 2655 bp in channel 2. The pWCF100 plasmid was obtained by IN-Fusion. The fragment size was verified by double restriction enzyme electrophoresis, such as Figure 1 As shown in B, channel 1 is the pWCF100 plasmid digestion product of 4425bp (vector backbone) and 721bp (EGFP gene fragment). Using pWCF100 as a template, the EGFP gene was amplified by PCR to verify the correctness of the plasmid, as shown in Figure 1 As shown in C, channel 1 is a 721 bp EGFP gene fragment. The plasmid sequence was verified by Sangon Company.

[0070] 1.3.1.2 Cloning and identification of the recombinant plasmid pWCF100S-EGFP in Top10: Using pWCF100-EGFP as a template, PCR was performed to obtain a 4392 bp linear DNA (the full-length vector sequence of pWCF100-EGFP). Figure 2 As shown in A, channel 1 is 4392 bp. The linear DNA was self-ligated by homologous recombination to obtain the pWCF100S-EGFP plasmid. The double enzyme digestion verification results are shown in Figure 2 As shown in B, channel 1 is 3651 bp (vector backbone) and 721 bp (EGFP gene fragment). The plasmid sequence was confirmed to be correct by Bio-Technology Corporation.

[0071] 1.3.1.3 Cloning and verification of the recombinant expression plasmid pWCF101S-EGFP in Top10: PCR was performed using pWCF100S-EGFP as a template to obtain a 4089 bp linear DNA (pWCF100S-EGFP vector sequence). The results are shown in Figure 3 A. PCR was performed using pMG36e as a template to obtain a 212 bp linear DNA (P32 promoter sequence). The results are shown in Figure 3 B. The pWCF101S-EGFP prokaryotic expression plasmid was obtained by IN-Fusion, and the fragment size was verified by double restriction enzyme electrophoresis. The double restriction enzyme digestion results of the recombinant plasmid pWCF101S-EGFP in Top10 are shown in Figure 3As shown in C, channel 1 is 3548 bp (vector backbone) and 721 bp (EGFP gene fragment); sequencing by Bioengineering Corporation confirmed that the plasmid sequence was correct.

[0072] 1.3.1.4 Verification of the recombinant expression plasmid pWCF101S-EGFP in NC8: The recombinant expression plasmid pWCF101S-EGFP was transferred into Lactobacillus plantarum NC8 to obtain lactic acid bacteria NC8-pWCF101S-EGFP. The plasmid was extracted and the EGFP gene was amplified by PCR. The results are shown in Figure 3 As shown in D, channel 1 is 721 bp (EGFP gene fragment). The plasmid sequence was confirmed to be correct by Bio-Technology Corporation.

[0073] 1.3.2 Construction of empty vector plasmid pWCF101S-EE: 1.3.2.1 Cloning and verification of Top10 empty vector plasmid pWCF101S-EE: PCR was performed using pWCF101S-V1 as template to obtain 3660 bp linear DNA. Figure 4 As shown in A, channel 1 is 3660 bp (without the EGFP vector sequence). The pWCF101S-EE plasmid was obtained by IN-Fusion. The double restriction enzyme digestion verification result of Top10-pWCF101S-EE empty vector is shown in Figure 4 As shown in B, channel 1 is 3629 bp (empty vector sequence). The plasmid sequence was confirmed to be correct by Bio-Technology Corporation.

[0074] 1.3.2.2 NC8-pWCF101S-EE empty vector verification: The empty vector plasmid pWCF101S-EE was transferred into Lactobacillus plantarum NC8 to obtain NC8-pWCF101S-EE. The plasmid was extracted and PCR amplified. The results are shown in Figure 4 As shown in C, channel 1 is 222 bp (tag sequence). The plasmid sequence was confirmed to be correct by Bio-Technology Corporation.

[0075] 1.3.3 Verification of protein expression of new vector 1.3.3.1 Detection of protein secretion expression level of pWCF101S-EGFP in Top10: Western-blot detection was performed using EGFP primary antibody. After pWCF101S-EGFP was transferred into Top10, it constitutively secreted and expressed the EGFP reporter protein fused with Flag, which was about 31.8KDa. Figure 5 A and M in the middle are protein markers, lane 1 is the Top10-pWCF101S-EE empty vector control group; lane 2 is the Top10-pWCF101S-EGFP group.

[0076] 1.3.3.2 Detection of protein expression level of pWCF101S-EGFP in Top10 bacteria: Verification of protein expression level of pWCF101S-EGFP plasmid in colon sediment (Flag antibody) Figure 5 B. Verification of protein expression of pWCF101S-EGFP plasmid in the supernatant of large intestine (EGFP antibody) Figure 5 Middle C; M is a protein marker, lane 1 is the Top10-pWCF101S-EGFP group, and lane 2 is the Top10-pWCF101S-EE empty vector control group.

[0077] 1.3.3.3 Verification of protein expression of the new vector pWCF101S-EGFP in Lactobacillus plantarum NC8: Western-blot test showed that the new functional lactic acid bacteria NC8-pWCF101S-EGFP expressed a secretory protein of about 31.8 KDa. Verification of protein expression of pWCF101S-EGFP plasmid in the supernatant of NC8 lactic acid bacteria (Flag antibody) Figure 5 As shown in D, M is a protein marker, lane 1 is the NC8-pWCF101S-EE empty vector control group, lane 2 is the NC8-pWCF101S-EGFP (4h) group, lane 3 is the NC8-pWCF101S-EGFP (6h) group, and lane 4 is the NC8-pWCF101S-EGFP (8h) group. The protein expression of pWCF101S-EGFP plasmid in the supernatant of NC8 lactic acid bacteria was verified (EGFP antibody) as shown in Figure 5 E. M is a protein marker, Lane 1 is the NC8-pWCF101S-EE empty vector control group, Lane 2 is the NC8-pWCF101S-EGFP (4h) group, Lane 3 is the NC8-pWCF101S-EGFP (6h) group, and Lane 4 is the NC8-pWCF101S-EGFP (8h) group.

[0078] 1.3.4 Verification of plasmid stability and expression stability of pWCF101S-EGFP plasmid in NC8 lactic acid bacteria 1.3.4.1 Verification of plasmid stability of pWCF101S-EGFP plasmid in NC8 lactic acid bacteria: The bacteria from the first to the seventh generation were cultured, and the plasmids were re-extracted. The recombinant expression plasmid pWCF101S-EGFP was used as a template for PCR amplification and identification. The results showed that the band size was 721bp (EGFP gene fragment) and there was no significant change compared with the first generation plasmid. The results proved that the plasmid had good stability. PCR verification was as follows Figure 6 Middle A.

[0079] 1.3.4.2 Verification of protein expression stability of pWCF101S-EGFP plasmid in NC8 lactic acid bacteria: The bacteria from the first to the seventh generation were cultured and samples were prepared. The Western-blot test showed that the band size was 31.8kd (EGFP reporter protein fused with Flag), which had no significant change compared with the first generation protein sample. The results showed that the plasmid protein expression was stable. Western-blot verification showed that Figure 6 Middle B.

[0080] The above is only a preferred embodiment of the present invention, and those skilled in the art may modify or replace it with an equivalent. Any modification, equivalent replacement and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A stable lactic acid bacteria secretory expression vector, characterized in that: It includes a replication system, a resistance screening system and a target gene expression system; wherein the replication system includes a lactic acid bacteria replicon sequence and an Escherichia coli replicon sequence; the resistance screening system includes a screening marker gene and its regulatory elements; the target gene expression system includes a secretion signal peptide sequence, a cloning site and a terminator sequence; Wherein, the stable lactic acid bacteria secretion expression vector is selected from any one of the following: (1) A basic vector comprising the replication system, resistance screening system and target gene expression system; (2) a self-ligating vector obtained by self-ligating the basic vector; (3) A constitutive vector that additionally integrates the P32 promoter sequence on the basis of the self-ligating vector.

2. The stable lactic acid bacteria secretory expression vector according to claim 1, characterized in that: The selection marker gene is the ermB gene, and the regulatory element includes an ermB promoter sequence, an ermB leader peptide sequence and an ermB terminator sequence; The cloning site includes Nco I and Bam HI restriction site; The terminator sequence is a cbh terminator sequence.

3. The stable lactic acid bacteria secretory expression vector according to claim 2, characterized in that: The nucleotide sequence of the secretion signal peptide is shown in SEQ ID NO: 1, and / or the nucleotide sequence of the lactic acid bacteria replicon is shown in SEQ ID NO: 2, and / or the nucleotide sequence of the Escherichia coli replicon is shown in SEQ ID NO: 3, and / or the nucleotide sequence of the ermB gene is shown in SEQ ID NO: 4, and / or the nucleotide sequence of the ermB promoter is shown in SEQ ID NO: 5, and / or the nucleotide sequence of the ermB leader peptide is shown in SEQ ID NO: 6, and / or the nucleotide sequence of the ermB terminator is shown in SEQ ID NO: 7, and / or the nucleotide sequence of the cbh terminator is shown in SEQ ID NO:

8.

4. The stable lactic acid bacteria secretory expression vector according to claim 1, characterized in that: When the stable lactic acid bacteria secretory expression vector is the constitutive vector, the nucleotide sequence of the P32 promoter is shown in SEQ ID NO:

9.

5. The stable lactic acid bacteria secretory expression vector according to claim 1, characterized in that: It also comprises a marking system and / or a verification system; wherein the marking system comprises a 3xFLAG tag, and the verification system comprises an SP6 promoter and an EGFP gene.

6. The stable lactic acid bacteria secretory expression vector according to claim 5, characterized in that: The nucleotide sequence of the 3xFLAG tag is shown in SEQ ID NO: 10; and / or the nucleotide sequence of the SP6 promoter is shown in SEQ ID NO: 11; and / or the nucleotide sequence of the EGFP gene is shown in SEQ ID NO:

12.

7. A method for constructing a stable lactic acid bacteria secretory expression vector according to any one of claims 1 to 6, characterized in that: The method comprises the steps of constructing a base vector, comprising: Obtaining a DNA fragment of a replication system, wherein the replication system includes a lactic acid bacteria replicon sequence and an Escherichia coli replicon sequence; Obtaining DNA fragments of resistance screening system, including screening marker genes and their regulatory elements; Obtain the DNA fragment of the target gene expression system, including the secretion signal peptide sequence, cloning site and terminator sequence; Assembling the above DNA fragments into a single vector by seamless cloning method to form the basic vector; Optionally, when constructing the basic vector, the method further includes obtaining a DNA fragment of a marking system and / or a DNA fragment of a verification system, and introducing the obtained DNA fragment into the basic vector by a seamless cloning method; And / or, the method comprises the step of constructing a self-ligating vector, comprising: Performing PCR amplification using the basic vector as a template to obtain a DNA fragment containing the full-length sequence of the basic vector; Self-ligating the DNA fragment containing the full-length sequence of the basic vector by a seamless cloning method to obtain the self-ligating vector; And / or, the method comprises the step of constructing a constitutive vector, comprising: Performing PCR amplification using the self-ligating vector as a template to obtain a first DNA fragment; obtaining a second DNA fragment containing a P32 promoter sequence; The first DNA fragment and the second DNA fragment are assembled into a single vector by a seamless cloning method to form the constitutive vector.

8. Use of the stable lactic acid bacteria secretory expression vector according to any one of claims 1 to 6 in constructing recombinant lactic acid bacteria.

9. Use of the stable lactic acid bacteria secretory expression vector according to any one of claims 1 to 6 in expressing foreign genes.

10. An engineered strain containing the stable lactic acid bacteria secretion expression vector according to any one of claims 1 to 6.

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