New functional lactic acid bacteria expressing ETEC antigens and preparation method and application thereof

Abstract

The present invention relates to the field of microorganisms, and specifically to providing a new functional lactic acid bacteria expressing ETEC antigens, and a preparation method and application thereof. The recombinant Lactobacillus plantarum expressing ETEC antigens uses Lactobacillus plantarum NC8 as a host bacterium and a pPG612.1 expression vector, wherein the pPG612.1 expression vector contains the FaeC gene or LngC gene of ETEC. The present invention innovatively integrates the FaeC and LngC genes of ETEC into Lactobacillus plantarum NC8 for expression, and can activate the intestinal mucosal immune response by oral administration, which can not only directly counteract the intestinal damage caused by ETEC and protect the intestinal villus structure, but also can regulate the intestinal microenvironment, stimulate the body to produce a systemic immune response, improve the body's ability to resist ETEC infection, reduce the morbidity and mortality of ETEC infection, and thus improve the economic benefits of the pig farming industry.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a novel functional lactic acid bacterium expressing ETEC antigens, and a preparation method and application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Enterotoxigenic Escherichia coli (ETEC) infection is a major challenge facing the current pig farming industry, especially for piglets in the lactation and weaning periods. ETEC binds to the specific receptors of the small intestinal epithelial cells through its surface-specific pili, releases enterotoxins after colonization in the intestine, destroys the intestinal mucosa, causes a large loss of water and electrolytes, and causes severe diarrhea. According to surveys, in large-scale farms, diarrhea caused by ETEC infection accounts for about 26.4% of piglet diarrhea cases, causing huge economic losses to the farming industry.

[0004] At present, the prevention and treatment of ETEC infection faces many technical bottlenecks. Traditional antibiotic treatment methods have significant limitations: ETEC is prone to drug resistance, resulting in reduced treatment effectiveness; premature use of antibiotics will not only affect the growth and development of piglets, but also interfere with the normal colonization of their intestinal symbiotic flora. In terms of immune protection, the passive immune protection obtained by piglets through breast milk is relatively limited, and the level of maternal antibodies will drop rapidly after weaning, making piglets extremely susceptible to ETEC infection. Existing vaccine technology also has many shortcomings: there is currently a lack of effective vaccines that can be widely used. Traditional vaccines mainly stimulate systemic immune responses, have unsatisfactory local intestinal immune effects, and are easily interfered with by maternal antibodies, which affects the immune effect. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a safe and effective prevention and treatment scheme in the field of ETEC (Enterotoxigenic Escherichia coli) infection prevention and control, especially for diarrhea problems in piglets and other livestock and poultry. At present, ETEC infection prevention and control mainly relies on antibiotic treatment, which not only easily leads to drug resistance problems, but also affects animal growth and development and intestinal flora balance. Although traditional vaccines can prevent ETEC infections, they are still insufficient in terms of immune protection effect and ease of use. Therefore, it is urgent to develop a new and sustainable ETEC prevention and control technology.

[0006] To solve the above problems, the specific objectives of the present invention include: first, to provide an anti-ETEC functional lactic acid bacteria constructed by genetic engineering means, which can express ETEC specific antigen proteins (FaeC or LngC), thereby avoiding the use of antibiotics and reducing the risk of drug-resistant strains. Second, to provide a composition or product that can prevent and / or treat ETEC infection, immunomodulation, anti-inflammation and protect the intestine. The composition or product can activate intestinal immune response, regulate inflammatory response, protect intestinal structure, etc., enhance the host's intestinal immune defense ability, and effectively reduce the risk of ETEC infection. Third, to provide a disease prevention and control method suitable for large-scale breeding. Because lactic acid bacteria have the characteristics of high safety, acid and bile resistance, etc., they are suitable for long-term use, can form a sustainable disease prevention plan, and effectively improve breeding benefits. Fourth, to provide a method for preparing the above-mentioned functional lactic acid bacteria and its application in preventing and / or treating ETEC infection, immunomodulation, anti-inflammation and intestinal protection.

[0007] By achieving the above objectives, the present invention not only provides a new technical solution for ETEC infection prevention and control, but also reduces the use of antibiotics, reduces environmental pollution, and promotes the green and sustainable development of the aquaculture industry.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect of the present invention, a new functional lactic acid bacterium expressing ETEC antigens is provided, wherein the new functional lactic acid bacterium expressing ETEC antigens uses Lactobacillus plantarum NC8 as a host bacterium and pPG612.1 as an expression vector, wherein the pPG612.1 expression vector contains the ETEC fimbriae subunit gene FaeC or the outer membrane protein subunit gene LngC (hereinafter referred to as FaeC gene or LngC gene).

[0010] In an embodiment of the present invention, the novel functional lactic acid bacteria expressing ETEC antigens express and produce ETEC pili antigen FaeC or outer membrane protein LngC (hereinafter referred to as FaeC protein or LngC protein). In some embodiments of the present invention, the nucleotide sequence of the FaeC gene is shown in SEQ ID NO: 1. In some embodiments of the present invention, the nucleotide sequence of the LngC gene is shown in SEQ ID NO: 2. In some embodiments of the present invention, the amino acid sequence of the FaeC protein is shown in SEQ ID NO: 3. In some embodiments of the present invention, the amino acid sequence of the LngC protein is shown in SEQ ID NO: 4.

[0011] In the second aspect of the present invention, a composition is provided, which comprises the novel functional lactic acid bacteria expressing ETEC antigens described in the first aspect. In some embodiments of the present invention, the composition comprises a combination of the following recombinant Lactobacillus plantarum: a recombinant Lactobacillus plantarum with Lactobacillus plantarum NC8 as a host bacterium, pPG612.1 as an expression vector, and the pPG612.1 expression vector comprising the FaeC gene of ETEC, and a recombinant Lactobacillus plantarum with Lactobacillus plantarum NC8 as a host bacterium, pPG612.1 as an expression vector, and the pPG612.1 expression vector comprising the LngC gene of ETEC.

[0012] In the third aspect of the present invention, a product is provided, comprising the novel functional lactic acid bacteria expressing ETEC antigens described in the first aspect or the composition described in the second aspect. In an embodiment of the present invention, the product is a probiotic preparation, a pharmaceutical preparation or a feed additive.

[0013] In some embodiments of the present invention, the pharmaceutical preparation is a biological preparation. In some embodiments of the present invention, the biological preparation is a vaccine, in particular an oral live vector vaccine. In some embodiments of the present invention, the product or composition comprises at least 1×10 9 CFU / mL of recombinant Lactobacillus plantarum.

[0014] In the fourth aspect of the present invention, a method for constructing the new functional lactic acid bacteria expressing ETEC antigens as described in the first aspect is provided, which comprises: obtaining the FaeC gene or LngC gene fragment of ETEC; constructing the gene fragment into the pPG612.1 expression vector; and introducing the pPG612.1 expression vector containing the gene fragment into Lactobacillus plantarum NC8.

[0015] In some embodiments of the present invention, the gene fragment is obtained by PCR in step (1). In some embodiments of the present invention, the gene fragment is digested and connected with the pPG612.1 expression vector to obtain a recombinant expression vector. In some embodiments of the present invention, the expression vector is introduced into Lactobacillus plantarum NC8 by electroporation in step (3).

[0016] In some embodiments of the present invention, the construction method further includes a verification step: detecting the integration of the target gene and the expression of the target protein. Specifically, in one embodiment of the present invention, the construction method includes the following steps: (1) obtaining the FaeC gene or LngC gene fragment of ETEC by PCR; (2) cloning the gene fragment into the pEasyBlunt vector; (3) double-digesting the pEasyBlunt recombinant vector and the pPG612.1 vector; (4) connecting the target gene fragment after double-digestion with the pPG612.1 vector to obtain a recombinant expression vector; (5) transforming the recombinant expression vector into Escherichia coli MC1061 for amplification; (6) electrotransforming the amplified recombinant expression vector into Lactobacillus plantarum NC8.

[0017] In the fifth aspect of the present invention, there is provided a use of the novel functional lactic acid bacteria expressing ETEC antigens described in the first aspect, or the composition described in the second aspect, or the product described in the third aspect in the preparation of a product having at least one of the following uses: (1) a product for preventing or treating ETEC infection; (2) an immunomodulatory product; (3) an anti-inflammatory product; and (4) an intestinal protection product.

[0018] According to experiments of the present invention, the novel functional lactic acid bacteria expressing ETEC antigens or a combination thereof of the present invention can effectively prevent ETEC infection in piglets, which includes reducing ETEC infection rate, alleviating infection symptoms and improving growth performance.

[0019] According to the experiments of the present invention, the novel functional lactic acid bacteria expressing ETEC antigens or a combination thereof of the present invention can achieve immune regulation, especially immune enhancement, which includes activating T cells in the spleen (SP), mesenteric lymph nodes (MLN) and intestinal lamina propria lymph nodes (LPL) of piglets, activating T cell immune response, and promoting CD4 + IFN - γ+、CD4 + IL-4 + And the proliferation and differentiation of dendritic cells, increase the level of intestinal SIgA, and enhance mucosal immunity.

[0020] According to the experiments of the present invention, the new functional lactic acid bacteria expressing ETEC antigens or a combination thereof of the present invention have obvious anti-inflammatory ability, which is manifested in the ability to inhibit the expression of inflammatory factors (TNF-α, IL-6 and IL-1β), reduce the expression of inflammatory factor proteins in serum (reduce serum TNF-α level, IL-6 level and IL-1β level), improve intestinal inflammatory state and regulate immune balance.

[0021] According to the experiments of the present invention, the new functional lactic acid bacteria expressing ETEC antigens or a combination thereof of the present invention have an intestinal protective effect. According to the results of pathological sections, they can protect the intestinal villus structure, reduce tissue damage, maintain tissue integrity, and improve intestinal barrier function, thereby maintaining intestinal health.

[0022] In some embodiments of the present invention, the product is a probiotic preparation, a pharmaceutical preparation or a feed additive.

[0023] Compared with the prior art, the advantages of the present invention include: the recombinant Lactobacillus plantarum NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC constructed by the present invention and their combination show significant effects in preventing ETEC infection. These engineered bacteria can not only activate intestinal immune response, regulate inflammatory response, and protect intestinal structure, but also have the characteristics of safety, convenience, and economic feasibility, providing an innovative ETEC prevention and control solution for the pig industry.

[0024] In terms of immune activation, the recombinant Lactobacillus plantarum of the present invention can effectively activate T cells in LPL, SP and MLN of piglets, promote CD4 + IFN - γ + T Cell、CD4 + IL - 4 + The proliferation of T cells and dendritic cells increases the level of intestinal sIgA and enhances local mucosal immune function. In particular, the mixed application of two engineered bacteria can produce a synergistic effect, which is better than a single strain of engineered bacteria in terms of immune cells and inflammatory factors, providing better immune protection.

[0025] In terms of inflammation regulation and tissue protection, the recombinant Lactobacillus plantarum of the present invention significantly reduced the expression levels of pro-inflammatory factors such as TNF-α, IL-6, and IL-8 in intestinal tissues, as well as the protein levels of inflammatory factors such as TNF-α, IL-6, and IL-1β in serum. At the same time, these engineered bacteria can reduce intestinal damage caused by ETEC infection, protect the structural integrity of intestinal villi, and maintain intestinal barrier function, thereby effectively preventing ETEC infection.

[0026] In terms of application value, the present invention uses lactic acid bacteria as a carrier, avoids the risk of using antibiotics, does not contain endotoxins, and has good biosafety. Oral administration is adopted, which is easy to operate, does not require injection, can reduce stress response, and is particularly suitable for large-scale breeding applications. This convenient use method makes it have better practicality and promotion value.

[0027] In terms of economic benefits, the recombinant Lactobacillus plantarum of the present invention can significantly reduce the morbidity and mortality of ETEC infection and improve animal growth performance. At the same time, by reducing the use of antibiotics and disease losses, it can effectively reduce breeding costs and improve economic benefits. As an antibiotic substitute, the application of this technology is of great significance to promoting the green development of the pig industry.

[0028] In summary, the recombinant Lactobacillus plantarum developed by the present invention has significant advantages in immune activation, inflammation regulation, safety and economic benefits. As an innovative ETEC prevention and control method, it can not only effectively prevent diseases and improve breeding benefits, but also promote the green and sustainable development of the pig farming industry, and has important practical application value and industrial development significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0030] Figure 1 The gel electrophoresis results of gene cloning and recombinant plasmid construction are shown. Among them: (a) PCR amplification results of target gene, where M: DNA Marker DL 2000; 1: LngC; 2: FaeC. (b) PCR identification results of pEasyBlunt-FaeC and pEasyBlunt-LngC; where M: DNA Marker DL 2000; 1: FaeC; 2: LngC. (c) Double enzyme digestion identification results of pPG612.1; where M: DNA Marker DL 2000; 1: pPG612.1. (d) Double enzyme digestion identification results of pEasyBlunt-FaeC and pEasyBlunt-LngC; where M: DNA Marker DL 2000; 1: EasyBlunt-FaeC; 2: pEasyBlunt-LngC. (e) PCR verification of recombinant plasmids pPG-FaeC and pPG-LngC; where M: DNA Marker DL 2000; 1: negative control; 2: pPG-LngC; 3: pPG-FaeC. (f) Double restriction enzyme verification of recombinant plasmids pPG-FaeC and pPG-LngC; where M: DNA Marker DL 5000; 1: pPG-LngC; 2: pPG-FaeC.

[0031] Figure 2The results of PCR verification of the recombinant plasmid and Western blot verification of the recombinant strain are shown. Wherein: (a) PCR verification of pPG-FaeC and pPG-LngC plasmids in NC8; Wherein, M: DNA Marker DL 2000; 1: negative control; 2: pPG-FaeC; 3: pPG-LngC. (b) Western blot verification of NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC; Wherein, M: protein marker; 1: NC8 control; 2: NC8-pPG612.1-FaeC; 3: NC8-pPG612.1-LngC.

[0032] Figure 3 CD4 in piglet LPL + T Cell flow cytometry detection results.

[0033] Figure 4 CD4 in piglet LPL + IL-4 + T Cell flow cytometry detection results.

[0034] Figure 5 CD8 in piglet LPL + T Cell flow cytometry detection results.

[0035] Figure 6 CD21 in piglet LPL + DCs flow cytometry detection results.

[0036] Figure 7 CD4 in piglet SP + IFNγ + T Cell flow cytometry detection results.

[0037] Figure 8 CD4 in piglet SP + IL4 + T Cell flow cytometry detection results.

[0038] Fig. 9 CD4 in MLN of piglets + IFNγ + T Cell flow cytometry detection results.

[0039] Fig.10 The results of the detection of transcription levels of TNF-α, IL-6, IL10 and IL-8 in the small intestine of piglets are shown.

[0040] Fig.11The results of ELISA assays of TNF-α (A), IL-6 (B), IL-1β (C) in piglet serum and sIgA (D) in piglet feces after challenge are shown.

[0041] Fig.12 Shown are the histopathological images of the jejunum of piglets in the PBS group (A), ETEC group (B), MIX group (C), LngC group (D), FaeC group (E), and PPG group (F). DETAILED DESCRIPTION

[0042] The present invention is further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope thereof. Experimental methods for which specific conditions are not specified in the examples are generally carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise defined, all professional terms and scientific terms used in the present invention should have meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the present invention can be obtained through conventional routes and used in accordance with conventional methods or product instructions in the art. In addition, any content similar or equivalent to the methods or materials described can be applied to the methods of the present invention. The preferred embodiments and materials described in the present invention are for illustrative purposes only.

[0043] Example 1: Construction and verification of new functional lactic acid bacteria against ETEC

[0044] 1.1 Materials and methods

[0045] 1.1.1 Strains and plasmid vectors: The strains used in this example include plasmid cloning hosts E. coli MC1061 and protein expression vector L. plantarum strain NC8, which was preserved and provided by the Microecological Preparation Engineering Research Center of Jilin Agricultural University. The plasmids required for the experiment include: pPG-612, pEasyBlunt (both preserved and provided by the Microecological Preparation Engineering Research Center of Jilin Agricultural University) and pUC-FaeC, pUC-LngC (both purchased from Shenggong Bioengineering Co., Ltd.), all of which carry the target gene. Among them, the nucleotide sequence of the FaeC gene is shown in SEQ ID NO: 1; the nucleotide sequence of the LngC gene is shown in SEQ ID NO: 2.

[0046] 1.1.2 Main reagents: The reagents required for this example include: restriction endonucleases, DNA Markers (DL2000, DL5000) and 10× Loading Buffer were all purchased from TAKARA official website; BCA protein concentration determination kit was purchased from Bio-Tech Biotechnology Co., Ltd.; ECL developer and 80 kDa Prestained Protein Marker were purchased from ProteinTech; beef powder, yeast extract and trypsin were purchased from Beijing Solebow Technology Co., Ltd.; SDS-PAGE loading buffer was purchased from Yazyme Biotechnology Co., Ltd.; SDS-PAGE fast electrophoresis buffer, Western Blot ice-free fast transfer buffer, and TBS and PBS buffers were all purchased from Wuhan Saiweier Technology Co., Ltd.; plasmid mini-extraction kit was purchased from Omega Biotechnology Co., Ltd.; T4 DNA ligase was purchased from Quanshijin Biotechnology Co., Ltd.

[0047] 1.1.3 The instruments required for this example include: a PCR instrument and an inverted fluorescence microscope DMi8 purchased from Leica, Germany; a gel imaging analysis system and an electroporation system purchased from BIO-RAD; a PCR instrument, a centrifuge and a pipette purchased from Eppendorf, Germany; a biochemical incubator HERACELL 240i purchased from Thermo Scientific, USA; a fully automatic high-pressure sterilizer purchased from Sigma, Germany; a 2 mm electric shock cup purchased from BIO-RAD, USA; and a metal bath purchased from TANGEN.

[0048] 1.1.4 Reagents and configurations required for the test

[0049] (1) Culture medium configuration

[0050] LB medium formula: weigh 10.0 g peptone, 10.0 g yeast extract and 5.0 g sodium chloride, dissolve in double distilled water and dilute to 1 L, dispense into conical flasks and test tubes, sterilize at 121°C for 15 min. Store at room temperature after sterilization. When preparing solid LB medium, add 15 g bacteriological agar powder per L of medium, sterilize under high pressure, and pour into bacterial culture dishes after appropriate cooling.

[0051] MRS liquid culture medium formula: weigh 10.0 g beef extract, 10.0 g peptone, 20.0 g glucose, 5.0 g sodium acetate trihydrate, 0.05 g yeast extract, 2.0 g ammonium citrate, 2.0 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, add 1 mL Tween-80, dissolve with double distilled water and dilute to 1 L, dispense into conical flasks and test tubes, sterilize at 121°C for 15 min. Store at room temperature after sterilization. When preparing solid LB culture medium, add 15 g bacteriological agar powder per L of culture medium, sterilize under high pressure, and pour into bacterial culture dishes after appropriate cooling.

[0052] (2) TAE (50×) nucleic acid electrophoresis buffer preparation formula: weigh 242 g Tris base, add 57.1 mL acetic acid and 100 mL EDTA (0.5 mol / L), dissolve with double distilled water and make up to 1 L. The solution needs to be fully dissolved and stored at room temperature.

[0053] (3) Agarose gel (1%) preparation formula: Weigh 1 g agarose and add it to 100 mL 1×TAE buffer. Heat until completely dissolved. After cooling to about 60°C, add 5 μL nucleic acid dye and mix thoroughly. Pour the gel and let it solidify at room temperature for later use.

[0054] (4) Preparation of washing buffer: Add 10 mL potassium acetate (1 mol / L, pH 7.0), weigh 11.8 g calcium chloride, 4.0 g manganese chloride and 2.0 g magnesium chloride, dissolve them in double distilled water and make up to 1 L. Adjust the pH value of the solution to 6.4, filter and sterilize using a 0.45 μm pore size disposable filter, and then aliquot and store at 4°C for later use.

[0055] (5) Preparation of storage buffer: Add glycerol to the washing buffer to a final concentration of 25% (V / V). Use a disposable Nalgene filter (0.45 μm pore size) and store at 4°C after aliquoting.

[0056] (6) Preparation of NC8 competent cells and washing buffer: First, prepare 0.5 mol / L EDTA, filter and add to solution I, and store at 4°C for later use. The configurations of solution I and solution II are as follows:

[0057] Preparation of solution I: Weigh 34.23 g sucrose, add 20 mL glycerol, dissolve and dilute to 1 L with double distilled water, sterilize at 121℃ for 15 min, and store at 4℃ for later use.

[0058] Preparation of solution II: Take 1 L of solution I, add filtered 0.5 mol / L EDTA to a final concentration of 0.05 mol / L, mix well and store at 4°C for later use.

[0059] (7) Preparation of Western Blot separation gel (10%): Take 3.3 mL 30% Acr / Bis (29:1), 2.5 mL 1.5M Tris-HCl (pH 6.8), 100 μL 10% SDS, 100 μL 10% PAGE gel coagulant, 10 μL PAGE gel accelerator, add 4 mL double distilled water, mix gently and pour the gel immediately.

[0060] (8) Preparation of Western Blot concentrated gel (5%): Take 0.83 mL 30% Acr / Bis (29:1), 0.625 mL 1M Tris-HCl (pH 6.8), 50 μL 10% SDS, 75 μL 10% PAGE gel coagulant, 7.5 μL PAGE gel accelerator, add 3.42 mL double distilled water, mix gently and pour the gel immediately.

[0061] (9) Formula for preparing 80% glycerol: Take 80 mL of glycerol and dilute to 100 mL with double distilled water. Stir thoroughly and seal the bottle. Autoclave at 121°C for 15 min. After the solution cools, store at 4°C for later use.

[0062] 1.2 Experimental methods

[0063] 1.2.1 Construction and identification of engineered bacteria

[0064] 1.2.1.1 Acquisition and synthesis of target genes: Based on the ETEC FaeC gene sequence (AF450247.2) and the ETEC Lngc gene sequence (LR883052) reported in the GenBanK database, primers were designed using Primer Premier 5.0 software. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd. Primer list: FaeC-F (SEQ ID NO: 5), FaeC-R (SEQ ID NO: 6), Lngc-F (SEQ ID NO: 7), Lngc-R (SEQ ID NO: 8).

[0065] 1.2.1.2 Preparation of template DNA: Take out the ETEC strain from the -80℃ refrigerator, streak it on LB solid medium, and culture it at 37℃ for 10h. Pick a single colony and place it in LB liquid medium without resistance, culture it at 37℃ for 12h, boil it in boiling water for 10min, centrifuge it at 10,000rpm at 4℃ for 15min, take the supernatant as DNA template, and store it at -20℃.

[0066] 1.2.1.3 Construction of pEasyBlunt-FaeC and pEasyBlunt-LngC cloning vectors: The FaeC and LngC fragments obtained by PCR amplification were cloned into the pEasyBlunt vector to obtain pEasyBlunt-FaeC and pEasyBlunt-LngC recombinant plasmids, respectively. After PCR amplification, the recombinant plasmids were identified by DNA sequencing.

[0067] 1.2.1.4 Vector and target gene cloning: Ppg612.1, pEasyBlunt-FaeC and pEasyBlunt-LngC were double-digested respectively. The digestion reaction system was as follows: 25 μL of plasmid was added with 2 μL of Nco I / Asc I and 2 μL of EcoR V / XhoI restriction enzymes, 5 μL of buffer, and 16 μL of double distilled water. After mixing, the mixture was reacted at 37°C for 6 h. 2 μL of the digestion product was taken for agarose gel electrophoresis (150 V, 20 min) to verify the digestion effect. After confirmation of the digestion, the gel was recovered.

[0068] 1.2.1.5 Connect the expression vector and the target gene sequence: Connect the recovered linear vector to the target sequence using T4 DNA ligase. Ligation reaction system: Take 2 μL of the linear vector recovery product, 5 μL of the target sequence recovery product, 1 μL of 10×T4 DNA ligase buffer and 1 μL of T4 DNA ligase, add 1 μL of double distilled water, mix well and react at 16°C overnight. Perform the transformation experiment the next day.

[0069] 1.2.1.6 E. coli Preparation of competent cells of MC1061: (1) Use an inoculation loop to streak MC1061 on the surface of LB solid culture medium and culture in a bacterial incubator at 37℃ for 14-16h. (2) The next day, pick a single colony of MC1061 and place it in 5mL LB culture medium and culture it in a shaking incubator at 37℃ for 14-16h. (3) Inoculate 1.5% of the MC1061 into 100mL LB and culture it in a shaking incubator at 37℃ until the OD600 is about 0.5 (2.5-3h). (4) Aliquot the bacterial solution into sterile 50mL centrifuge tubes, place on ice for 10min, centrifuge at 4℃, 5000 rpm for 10min, and discard the supernatant. (5) Use 10mL pre-cooled washing buffer to gently suspend the bacteria, place on ice for 10min, centrifuge at 4℃, 5000 rpm for 10min, and discard the supernatant. (6) Lightly suspend the cells with 10 mL of pre-cooled washing buffer, place on ice for 20 min, centrifuge at 4°C, 5,000 rpm for 10 min, and discard the supernatant. (7) Lightly suspend the cells with pre-cooled storage buffer, place on ice for 30 min. Dispense the above competent cells into pre-cooled 1.5 mL EP tubes at 50 μL / tube, freeze in liquid nitrogen, and store at -80°C. All the above operations were performed in a clean bench to prevent contamination.

[0070] 1.2.1.7 Transformation of ligation product: The ligation product was transformed into MC1061 competent cells by heat shock transformation method. The steps include: (1) Gently mix 5 μL ligation product with 50 μL competent cells and incubate on ice for 30 min; (2) Preheat the water bath to 42°C, heat shock for 90 s, and cool on ice for 2 min; (3) Add 400 μL LB liquid culture medium, incubate at 37°C bacterial shaker at 200 rpm for 2 h; (4) Centrifuge at 5 000 rpm for 5 min, take 100 μL and plate; (5) Incubate in a 37°C bacterial incubator, place upright for 30 min, then invert for 12-14 h. After a single colony appears, pick a single colony and place it in a 5 mL LB liquid culture medium tube, incubate at 37°C shaker, and save the strain for subsequent experiments.

[0071] 1.2.1.8 Extraction of recombinant expression plasmid and primer synthesis in MC1061: Use a plasmid extraction kit to extract the plasmid as follows. (1) Inoculate E. coli carrying the plasmid into 5 mL LB (containing chloramphenicol) and culture at 37°C for 12-16 h. Take 5.0 mL of bacterial solution and centrifuge at 10,000×g for 1 min at room temperature to collect the bacteria. (2) Discard the culture medium. Add 250 μL Solution I / RNase A mixture, pipette to completely suspend the cells, add 250 μL Solution II, gently invert 4-6 times to mix, and the lysis reaction should not exceed 5 min. (3) Add 350 μL Solution III, gently invert 7-9 times until white flocs appear, and centrifuge at 10,000×g for 10 min. (4) Transfer the supernatant to a HiBind DNA binding column in a 2 mL collection tube, centrifuge at 10,000 × g for 1 min at room temperature, and discard the filtrate in the collection tube. (5) Return the binding column to the collection tube, add 500 μL HBBuffer, centrifuge as above, and discard the filtrate. (6) Return the column to the collection tube, add 700 μL DNA Wash Buffer, centrifuge as above, and discard the filtrate. Note: Before use, add anhydrous ethanol to the concentrated DNA Wash Buffer as indicated on the label. (7) Discard the filtrate and repeat step 6 once. (8) Discard the filtrate, return the column to the collection tube, centrifuge at 10,000 × g, and leave the column empty for 2 min to dry the column matrix. (9) Place the column in a 1.5 mL centrifuge tube, add 30-50 μL Elution Buffer to the column matrix, let stand for 2 min, and centrifuge at 10,000 × g for 1 min to elute the DNA. The extracted plasmid was stored at -20°C for PCR and restriction enzyme identification. Primer sequences and restriction enzyme sites: pPG612 plasmid restriction enzyme site Nco I (primer F) is shown in SEQ ID NO: 9, pPG612 plasmid restriction enzyme site Asc I (primer R) is shown in SEQ ID NO: 10.

[0072] 1.2.1.9 PCR identification of recombinant expression plasmid

[0073] PCR reaction system: Take 1 μL template plasmid, 1 μL forward primer (F) and 1 μL reverse primer (R), add 25 μL 2× PrimeSTAR® Mix and 22 μL double distilled water, centrifuge quickly and place on a PCR instrument for amplification reaction.

[0074] PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 60℃ annealing for 40 s, 72℃ extension for 4 min, 35 cycles; 72℃ final extension for 10 min; 4℃ hold. Take 10 μL of PCR product for agarose gel electrophoresis.

[0075] 1.2.1.10 Double enzyme digestion identification of pPG612.1-FaeC and pPG612.1-LngC recombinant expression plasmids: The double enzyme digestion reaction system is the same as the enzyme digestion reaction system in 1.2.1.4. Digest at 37℃ for 6h, take 2μL of the digestion product and perform agarose gel electrophoresis at 170V for 20min to compare the band sizes, using the same method as the instruction manual.

[0076] 1.2.1.11 Sequencing and identification of recombinant expression plasmids: The correct plasmid samples identified by PCR and enzyme digestion were sequenced and identified by Sangon Biotechnology Co., Ltd., and the plasmid map was drawn using SnapGen software.

[0077] 1.2.1.12 Preparation of competent cells of L. plantarum strain NC8: (1) Activation of bacterial strains: Inoculate NC8 strains into 5 mL of MRS medium and culture anaerobically in a 37°C incubator for 14-16 h. (2) Subculture: Inoculate 1% of the strain into 100 mL of MRS medium containing 2% GLy. Test the OD value every 2 h (visual inspection is possible, no frequent testing is required, the final OD value is 0.3-0.5). (3) Centrifugation: Divide into 50 mL sterile tubes, centrifuge at 4°C, 5,000 rpm for 10 min, discard the supernatant, and collect the cells. (4) Suspend the cells with 20 mL of solution I, incubate at 4°C, 5,000 rpm for 10 min, and collect the cells. (5) Wash once with 20 mL of solution II, at 4°C, 5,000 rpm for 10 min, and collect the cells. (6) Suspend the cells with 20 mL of solution I, place on ice for 15 min, and collect the cells at 4°C, 5,000 rpm for 20 min. (7) Repeat step 6 to thoroughly wash off the EDTA. (8) Suspend the cells with 1 mL of solution I, aliquot at 100 μl / tube, freeze in liquid nitrogen, and store at -80°C.

[0078] 1.2.1.13 Electroporation of recombinant expression plasmid: The recombinant plasmid that has been sequenced correctly is transformed into NC8 competent cells by electroporation. (1) Thaw NC8 competent cells on ice, add 10μL of plasmid to the competent cells, mix gently, and incubate on ice for 5 minutes. (2) Pre-cool the electroporation cup on ice in advance, transfer the plasmid competent mixture to the electroporation cup (try to avoid bubbles), and ice bath for 10 minutes. (3) Electroporation conditions: 2kV, 200Ω, 25µF. (4) After the electroporation is completed, quickly add 900μL of MRS culture medium (the culture medium is preheated at 37℃ in advance) and shake at 37℃, 200rpm, and culture for 60 minutes. (5) Take 100μL of bacterial solution and evenly spread it on the MRS resistance agar plate, culture it upright for 1 hour, and culture it inverted for 12-16 hours until a single colony can be seen. (6) Pick a single colony and place it in 10 mL of MRS resistance medium. Incubate in a shaker at 37°C, 200 rpm for 12-16 hours. Save the strain for subsequent testing.

[0079] 1.2.1.14 Identification of NC8 recombinant expression plasmid: Extract the plasmid from the engineering bacteria using the same method, and perform PCR identification on the recombinant plasmid using the same method as step 1.2.1.9.

[0080] 1.2.2 Verification of engineered bacterial protein expression

[0081] 1.2.2.1 Determination of the growth curve of engineered bacteria: After the engineered strain is activated, 100L is inoculated into 5mL MRS broth and shaken at 37℃ and 200rpm overnight. The next day, 1% inoculation amount is inoculated into 100mL MRS liquid culture medium and cultured at 37℃ and 200rpm. Take 2mL of bacterial solution every 2 hours to measure the OD600 value and record it. Continue to monitor for 24 hours and draw the strain growth curve through Prisme.

[0082] 1.2.2.2 Identification of engineered bacterial protein expression: Inoculate 100 μL of engineered bacteria into MRS broth and shake at 37°C 200 rpm overnight. The next day, inoculate 1% of the inoculation volume into 100 mL of MRS medium containing 2% lactose and culture overnight at 37% 200 rpm. Centrifuge and discard the supernatant, wash twice with sterile PBS, and resuspend in PBS buffer for later use. Determine the protein concentration by BCA method, take 400 μL of the resuspended bacterial solution and mix it evenly with 100 μL 5×SDS PAGE Buffer, and boil for 10 min. Centrifuge at 12,000 rpm for 1 min, collect the supernatant and store at -40°C.

[0083] 1.2.2.3 Western blot detection: Western blot technology was used to verify the expression of engineered bacterial proteins. The specific method is as follows: (1) After the gel plate is installed, use ultrapure water to check whether the gel plate is leaking. Check for leaks for 15 minutes. If the water level does not drop, continue; (2) Add the prepared 10% separation gel (lower layer gel) to the glass plate, seal it with anhydrous ethanol, wait for 20 minutes for solidification, discard the anhydrous ethanol, absorb the residual anhydrous ethanol with filter paper, and then add the concentrated gel (upper layer gel) to the electrophoresis comb and wait for solidification. After solidification, place the PAGE gel in the electrophoresis tank and add an appropriate amount of SDS-PAGE fast electrophoresis buffer; (3) Sample addition: Add 20μL of treated protein sample and 5μL of marker to the gel well; (4) Electrophoresis conditions: 200V (about 30min) bromophenol blue to the bottom edge of the glass plate to stop electrophoresis; (5) Transfer: Take out the gel and place it in the order of sponge, filter paper, PVDF membrane, gel, filter paper, and sponge from positive to negative. Gently remove the bubbles, place it in the transfer clip, and place it in ice together with the electrophoresis tank; (6) Transfer conditions: constant current 400mA, 30min; (7) Blocking: Place it in the blocking solution and shake it slowly at room temperature for 2h; (8) Primary antibody incubation: Add anti-His primary antibody (diluted with TBST) and incubate at 4℃ overnight. Rinse the PVDF membrane with TBST three times, 10min each time; (9) Secondary antibody incubation: Add enzyme-labeled secondary antibody (diluted with TBST) and shake it slowly on a shaker at room temperature for 1h. The PVDF membrane was rinsed with TBST three times, each time for 10 min; (10) the membrane was moistened with ECL luminescent solution and placed in the AI600 imaging system for exposure and observation of protein blot.

[0084] 1.3 Results

[0085] 1.3.1 Construction of engineered bacteria

[0086] 1.3.1.1 Obtaining and synthesizing target genes: Obtain FaeC and LngC gene sequences by PCR, such as Figure 1 As shown in (a), the sizes of FaeC and LngC gene fragments are 546 bp and 414 bp, respectively, which are consistent with the expected sizes.

[0087] 1.3.1.2 Construction and identification of pEasyBlunt-FaeC and pEasyBlunt-LngC cloning vectors: The two target gene sequences were finally obtained by fusion PCR and connected to the pEasyBlunt vector. FaeC and LngC were identified by PCR. The results are as follows: Figure 1 Middle (b) shows that expected size bands appeared at 546 bp and 414 bp.

[0088] 1.3.1.3 Verification of expression vector and target gene: Double restriction enzyme digestion detection was performed on the pPG612 vector plasmid and the target gene sequence on pEasyBlunt. Figure 1 As shown in (c) and (d), the target bands were visible at 4233 bp (c); 546 bp and 414 bp (d), which was consistent with the expected results.

[0089] 1.3.1.4 Identification of recombinant expression plasmid in MC1061

[0090] 1.3.1.4.1 PCR identification of recombinant expression plasmids: Using the suspected positive recombinant expression plasmids pPG-FaeC and pPG-LngC as templates, PCR amplification and identification were performed. The results were as follows: Figure 1 As shown in (e), the target bands at 718 bp and 586 bp are in line with the expected size.

[0091] 1.3.1.4.2 Double enzyme digestion identification of recombinant expression plasmids: Double enzyme digestion verification of recombinant expression plasmids pPG-LngC and pPG-FaeC was performed. The results are as follows Figure 1 As shown in (f), the target bands were visible at 718 bp and 586 bp, respectively, confirming that the target fragment had been successfully connected to the expression vector.

[0092] 1.3.1.4.3 Sequencing identification of recombinant expression plasmid: The recombinant plasmid was sequenced by Sangon Biotechnology (Shanghai) Co., Ltd. The sequencing results were correct and there was no base mutation2.

[0093] 1.3.1.5 PCR identification of recombinant expression plasmid in NC8

[0094] The target gene of the recombinant plasmid in NC8 was amplified by PCR to detect whether the recombinant plasmid was successfully transferred into NC8. Figure 2 As shown in (a), the target bands were visible at 718 bp and 586 bp, proving that the recombinant plasmid was successfully transferred into the vector bacteria NC8.

[0095] 1.3.2 Protein expression verification

[0096] 1.3.2.1 Growth curve determination: Growth curve determination and colony counts were performed on NC8, NC8-pPG612.1, NC8-pPG612.1-FaeC, and NC8-pPG612.1-LngC, and 24h OD was determined. 600 The growth curve was plotted using GraphPad Prism software. During the stable period, there was no significant difference between the engineered bacteria and the original bacteria.

[0097] 1.3.2.2 Western blot test results of engineered bacteria: Through Western blot test, specific proteins were observed at 51kDa and 40kDa, respectively. This proves that lactose MRS culture can successfully induce NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC to express corresponding proteins, and specific proteins were observed at 51kDa and 40kDa, respectively. Figure 2 As shown in (b).

[0098] Example 2: Evaluation of the immune effect of new functional lactic acid bacteria against ETEC.

[0099] 2.1 Materials and methods

[0100] 2.1.1 Test strains: New functional lactic acid bacteria NC8-pPG612.1, NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC (constructed in Example 1); ETEC was provided by the Microecological Preparation Engineering Research Center of Jilin Agricultural University.

[0101] 2.1.2 Experimental animals: 18 3-day-old suckling piglets.

[0102] 2.1.3 Main reagents: Nylon mesh (Solarbio); FBS red blood cell lysis buffer, DAPI stain, fetal bovine serum, Triton X-100 and BSA were purchased from Bio-Tech Biotechnology Co., Ltd.; 4% paraformaldehyde and RPMI-1640 culture medium were purchased from Biosharp; Anti-pig CD4, Anti-pig CD8 and Anti-pig IgA antibodies were purchased from BD (USA).

[0103] 2.1.4 Main instruments: The flow cytometer was purchased from BD Company, USA, and the automatic embedding machine and automatic paraffin sectioning machine were purchased from Leica.

[0104] 2.2 Experimental methods

[0105] 2.2.1 Plate count of engineered bacteria: (1) Take the strain from -80℃ and inoculate 1% of the engineered bacteria into 5mL MRS medium containing chloramphenicol at 200rpm and culture overnight. (2) Inoculate 1% of the revived strain into 200mL MRS medium containing chloramphenicol and culture at 37℃ in a shaking incubator until OD 600 The value is 1. (3) Dilute the cultured bacterial solution to 10 -5 , 10 -6 , 10 -7Take 100 μL of the diluted bacterial solution and spread it evenly on MRS solid culture medium. Do three replicates for each ladder and culture it anaerobically at 37°C for 24 h. Take the average value for colony counting.

[0106] 2.2.2 Experimental animal grouping and immunization scheme: The piglets were randomly divided into 6 groups, with 3 pigs in each group, namely PBS group (group A), ETEC group (group B), NC8-pPG612.1 (group C), NC8-pPG612.1-FaeC (group D), NC8-pPG612.1-LngC (group E), and engineered bacteria mixed group (group F).

[0107] The immunization scheme of each group was as follows: Group A was orally administered with 1 mL PBS as a control; Group B was orally administered with 1 mL ETEC; Group C was orally administered with 1 mL NC8-pPG612.1 (1×10 9 CFU / mL); Group D was orally administered with 1 mL NC8-pPG612.1-FaeC (1×10 9 CFU / mL); Group E was orally administered with 1 mL NC8-pPG612.1-LngC (1×10 9 CFU / mL); Group F was the engineered bacteria mixed immunization group, and 1 mL of engineered bacteria mixed solution (total bacterial count of 1×10 9 CFU / mL, where NC8-pPG612.1, NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC were mixed in equal amounts).

[0108] Immunization and virus attack scheme: OD of engineered bacteria was obtained by plate counting 600 =1, and adjust the concentration of bacterial solution for immunization accordingly. Each piglet was gavaged with 1×10 9 CFU / mL, immunized once every 1 day, for a total of 7 times. After the last immunization, the virus was challenged by gavage every 1 day, and the challenge dose was 1 mL ETEC per kg body weight (5.34×10 7 CFU / mL). The piglets were observed for 7 days, and their body weights were counted and their weight growth rate was calculated daily. After 7 days, their body weights were weighed and their blood was collected. All piglets were then killed and samples were collected.

[0109] 2.2.3 Serum collection: All piglets were killed 7 days after the last immunization. First, fix the front and rear limbs of the piglets, and use a 5mL syringe to pierce the skin at a 45° angle at the left clavicular fossa (jugular vein groove) to collect blood. Collect blood and 5mL centrifuge tubes, let stand in a 37℃ incubator for 1 hour, precool the centrifuge at 4℃, 4 000rpm, 15min, collect the supernatant, and store it in a -80℃ refrigerator for subsequent experiments.

[0110] 2.2.4 Collection of fecal supernatant: Piglets were killed by bleeding from the jugular vein. After killing, the abdominal cavity was opened and the feces from the jejunum were collected and placed in a 5 mL centrifuge tube. Three volumes of PBS were added according to the volume of the feces, and protease inhibitors were added. The centrifuge was precooled at 4°C and centrifuged at 4000 rpm for 15 min. The fecal supernatant was collected and stored in a -80°C refrigerator for subsequent experiments.

[0111] 2.2.5 Flow cytometry: spleen (SP), mesenteric lymph nodes (MLN) and intestinal lamina propria lymph nodes (LPL) were collected from piglets.

[0112] 2.2.5.1 Preparation of single cell suspension: (1) Place a 200-mesh copper mesh in a 35-nm cell culture dish, add 1 mL of complete culture medium, and incubate at 4°C for use. (2) Place tissues (SP, MLN, LPL) in the copper mesh, grind with the tail of a 1 mL syringe, grind the tissue into a homogenate, and pipette the tissue stock solution filtered through the copper mesh into a 1.5 mL centrifuge tube. (3) Add 1 mL of pre-cooled red blood cell lysis buffer to the SP cell suspension, mix gently, place on ice for 5 min, add 500 μL of PBS, centrifuge at 4°C, 500 × g for 5 min, discard the supernatant, gently suspend in PBS, centrifuge again, and select the precipitate with PBS after centrifugation. (4) Suspend the SP, MLN, and LPL cell pellets with 1 mL of PBS, centrifuge at 4°C, 500 × g for 5 min, discard the supernatant, and gently suspend in 1 mL of PBS buffer. (5) Dilute each group of cells with PBS buffer in multiple ratios, count the cells after dilution using a red blood cell counting plate, and calculate the concentration of the stock solution based on the number of cells. (6) Take the required volume of cell stock solution, suspend it lightly with 1 mL of PBS, centrifuge it at 4°C, 500 × g for 5 min, discard the supernatant, resuspend it with 100 μL of PBS, and store it at 4°C for later use.

[0113] 2.2.5.2 DCs detection: (1) Add CD14C-percp-cy5.5 (40-fold dilution) and CD21-percp-cy5.5 (40-fold dilution) to 100 μL of single cell suspension, add 20 μL to each sample and gently pipette to mix, incubate at 4°C in the dark for 30 min. (2) After incubation, add 1 000 μL PBS buffer, centrifuge at 4°C, 500×g for 5 min, and discard the supernatant. (3) Add 200 μL PBS buffer to resuspend the cells, filter the cell solution into the flow tube using a nylon mesh, add 100 μL PBS buffer to mix, and store at 4°C for detection.

[0114] 2.2.5.3 B cell detection: (1) Add 10 μL of CD11 (35-fold dilution) antibody to 100 μL of single cell suspension, gently pipette to mix, and incubate at 4°C in the dark for 30 min. (2) After incubation, add 500 μL of PBS, centrifuge at 4°C, 1 000 rpm for 5 min, and discard the supernatant. (3) Add 80 μL of Fixation / Permeabilization to the precipitate to lightly suspend the cells, and incubate at 4°C in the dark for 45 min. (4) After incubation, add 150 μL of Permeabilization wash solution, centrifuge at 4°C, 500×g for 5 min, and discard the supernatant. (5) Add 10 μL of IgA-FITC (40-fold dilution) antibody, gently pipette to mix, and incubate at 4°C in the dark for 45 min. (6) After incubation, add 200 μL of permeabilization solution, centrifuge at 4°C, 500 × g for 5 min, discard the supernatant, and add 200 μL of PBS. (7) Use a nylon mesh to filter the cell solution into a flow tube, add 100 μL of PBS, and store at 4°C until detection.

[0115] 2.2.5.4 T cell detection: (1) Add CD3-APC (40-fold dilution), CD4-APC (40-fold dilution), and CD8-PE (35-fold dilution) to 100 μL of single cell suspension, mix the three antibodies, add 30 μL to each sample, gently pipette to mix, and incubate at 4°C in the dark for 45 min. (2) After the reaction, add 150 μL PBS to terminate the reaction, centrifuge at 4°C, 500×g for 5 min, discard the supernatant, and add 200 μL PBS. (3) Use a nylon mesh to filter the cell solution into a flow tube, add 100 μL PBS to mix, and store at 4°C until detection.

[0116] 2.2.6 ELISA test

[0117] 2.2.6.1 Detection of sIgA levels in piglet feces: Take the supernatant of piglet feces frozen in a -80℃ refrigerator and use ELISA to detect the content of sIgA in piglet feces.

[0118] 2.2.6.2 Detection of inflammatory cytokine levels in piglet serum: The supernatant of piglet blood frozen at -80°C was collected, and ELISA was used to detect the levels of TNF-α, IL-6, and IL-1β in piglet feces.

[0119] 2.2.7 Detection of cytokine mRNA expression levels

[0120] 2.2.7.1 Extraction of total RNA from jejunum: Take out frozen jejunum tissue from -80℃ freezer and extract total RNA from small intestine using Trizol method. Including: (1) Take 50mg intestinal tissue and add 1mL Trizol, grind the tissue into homogenate using electric grinder, precool the centrifuge at 4℃, centrifuge the homogenized sample at 4℃, 12000 rpm, 10min, collect the homogenate supernatant and place it in 1.5mL centrifuge tube. (2) Let the sample stand at room temperature for 5min, add 0.2mL chloroform to each tube of supernatant sample, vortex for 15s, and let it stand at room temperature for 5min. (3) Centrifuge the supernatant sample at 4℃, 12000rpm, 15min, and separate the samples after centrifugation. RNA is in the upper aqueous phase at this time. Take the upper aqueous phase and place it in a 1.5mL centrifuge tube. Note: Do not suck up the white film. (4) Add 2 times the volume of isopropanol, mix by inverting the centrifuge tube, let stand at room temperature for 5 min, let stand on ice for 5 min, centrifuge at 4 ℃, 12,000 rpm, 10 min, and discard the supernatant. (5) Add 1 mL of pre-cooled 75% ethanol (DEPC water preparation) to wash, vortex to mix the sample solution, centrifuge at 4 ℃, 12,000 rpm, 5 min, and discard the supernatant. (6) Open the tube cap, place it on the operating table to dry naturally (do not dry it completely), add 30 μL of DEPC water, and place it in a 37 ℃ metal bath for 15 min to dissolve the RNA. Store in a -20 ℃ refrigerator for subsequent experiments.

[0121] 2.2.7.2 Reverse transcription of total RNA from small intestinal tissue: The extracted total RNA was reverse transcribed and the reverse transcription product was stored at -20°C for subsequent qPCR detection of transcriptional differences in intestinal inflammatory cytokines. The reverse transcription reagent used was the reverse transcription kit (containing gDNA enzyme) from Bio sharp, and the operation method was referred to the instruction manual.

[0122] The reverse transcription reaction was carried out in a 40 μL system, which contained: 16 μL 5× RT MasterMix, 4 μL 20× Oligo dT&Random Primer, 0.1-4 μg Total RNA, and finally made up to 40 μL with RNase Free H2O. The reaction conditions were 37°C for 15-30 min, 85°C for 5 min, and finally kept at 4°C.

[0123] 2.2.7.3 qPCR detection: GapDH was used as the qPCR internal reference gene. qPCR primers were designed according to the gene sequences of porcine TNF-α, IL-6, IL-8, and IL-10 in the NCBI database. The reaction system was referred to the instruction manual. Primer sequences: GapDH-F (SEQ ID NO: 11), GapDH-R (SEQ ID NO: 12), TNF-α-F (SEQ ID NO: 13), TNF-α-R (SEQ ID NO: 14), IL-6-F (SEQ ID NO: 15), IL-6-R (SEQ ID NO: 16), IL-8-F (SEQ ID NO: 17), IL-8-R (SEQ ID NO: 18), IL-10-F (SEQ ID NO: 19), and IL-10-R (SEQ ID NO: 20).

[0124] 2.2.8 Histopathological examination

[0125] 2.2.8.1 Tissue embedding and sectioning: (1) Fixation: Use 4% paraformaldehyde for fixation. Generally, dehydration can be started after fixation for more than 3 days, and at least 2 days. (2) Sampling: Use a razor blade to trim the two sides of the intestinal tube. (3) Dehydration: Use alcohol for gradient dehydration. 70% 3h-4h (3.5h), 80% 3h-4h (3.5h), 85% 3h-4h (3.5h), 90% 3h-4h (3.5h), 95%Ⅰ (overnight), 95%Ⅱ (2h), 100%Ⅰ (2h), 100%Ⅱ (3h). (4) Transparency: Set xylene Ⅰ and xylene Ⅱ, with a transparent time of 5 minutes each, until it is transparent like cooked meat skin. (5) Wax immersion and embedding: The wax immersion temperature is 58°C; the wax immersion time of wax 1, wax 2, and wax 3 is 30 minutes each. Place the tissue in wax 3 into the wax block tray of the embedding machine and prepare for embedding. (6) The slice thickness is 3 μm, and the slices are spread in a 42°C water bath. (7) Place the slices in an 80°C oven for 1 hour.

[0126] 2.2.8.2 HE staining: The dried sections were stained according to the following procedure: xylene treatment twice (8 min each); gradient alcohol hydration (100% alcohol twice, 1 min each; 95%, 80%, 70% alcohol, 1 min each); after washing with water, stain with hematoxylin for 5 min and wash with water; 0.5% hydrochloric acid alcohol differentiation for 5 s; dilute ammonia water to blue for 2 min and then wash with water; 0.5% eosin aqueous solution staining for 5 min; gradient alcohol dehydration (80% alcohol quickly lifted up and down several times, 95% alcohol for 1 min, 100% alcohol twice for 1 min each); finally xylene treatment twice (2 min each).

[0127] 2.2.9 Data statistics and analysis: Graphical Prism 10 software was used for data statistics and analysis. The results between the experimental groups were tested using One-way ANOVA, and the significance level was: ns ( P >0.05);*( P <0.05);**( P <0.01);***( P <0.001); ****( P <0.0001).

[0128] 2.3 Results

[0129] 2.3.1 Weight changes of immunized piglets after ETEC challenge: Weight changes were recorded from the first day of the first immunization until the piglets were killed 7 days after the challenge. The weight growth curve showed that the weight of the ETEC control group showed a significant downward trend. After the challenge, the weight growth trend of the LngC group, FaeC group and PBS group was significantly higher than that of the ETEC group. After 3 days of the challenge, the weight growth trend returned to the level before the challenge.

[0130] 2.3.2 Analysis of flow cytometry results: In order to detect the immune effect of engineered bacteria, this experiment immunized piglets with engineered bacteria by gavage. Seven days after the challenge, flow cytometry was used to detect various indicators of spleen (SP), mesenteric lymph nodes (MLN) and intestinal lamina propria lymphocytes (LPL) in piglets, and flow cytometry data analysis was performed using FlowJo v10.6.2, and data analysis and graphing were performed using GraphPad Prism 10.

[0131] 2.3.2.1 Effects of engineered bacteria on immune cells in piglet LPL

[0132] In order to detect the effect of engineered bacteria on helper T cells in piglet LPL, this experiment was conducted on CD4 + T Cell was tested and the results were as follows Figure 3 Compared with the ETEC group, there were no significant differences among the groups ( P >0.05). At the same time, this experiment was performed on CD4 + IL-4 + T Cell was tested and the results were as follows Figure 4 Compared with the ETEC group, the MIX group showed a very significant difference ( P <0.0001), the LngC group and the FaeC group showed certain differences compared with the ETEC group ( P <0.05). This study examined the CD8 + Cell was tested and the results were as follows Figure 5Compared with the ETEC group, the MIX group showed significant differences ( P< 0.05), while there was no statistical difference in the other prevention groups ( P> 0.05). In this experiment, flow cytometry was used to detect the expression of CD21 in LPL cells of engineered bacteria. + The expression levels of DCs, such as Figure 6 As shown, compared with the ETEC group, the MIX group showed a very significant difference ( P <0.001, followed by LngC ( P <0.05).

[0133] The above research results show that the engineered bacteria administered orally can effectively activate T cells and dendritic cells in the intestinal lamina propria lymphoid tissue of piglets. This process promotes the recognition and uptake of pathogens and their antigens by intestinal antigen-presenting cells, as well as the transmission of immune signals.

[0134] 2.3.2.2 Effect of engineered bacteria on immune cells in piglet SP: In order to detect the effect of engineered bacteria on helper T cells in piglet SP, CD4 + IFNγ + T Cells were tested. The results are as follows Figure 7 As shown, compared with the ETEC group, the MIX group showed a very significant difference ( P< 0.0001), followed by LngC and FaeC groups, which showed significant differences ( P< 0.01). At the same time, this experiment was conducted on CD4 + IL4 + Cell was tested. The results are as follows Figure 8 As shown, compared with the ETEC group, the MIX group showed a very significant difference ( P< 0.001), FaeC group showed significant difference ( P< 0.01), LngC group showed some differences ( P< 0.05). The above results show that intragastric administration of engineered bacteria can effectively induce the activation of immune cells in mouse SP.

[0135] 2.3.2.3 Effect of engineered bacteria on immune cells in piglet MLN: In order to detect the effect of engineered bacteria on helper T cells in piglet MLN, this experiment was conducted on CD4 + IFNγ + Cell was tested. The results are as follows Fig. 9 As shown, compared with the ETEC group, the LngC group and the MIX group showed extremely significant differences ( P< 0.0001), followed by the FaeC group ( P<0.01). The results showed that the engineered bacteria could induce cellular immune response in mesenteric lymph node swelling of piglets.

[0136] 2.3.3 Detection of inflammatory cytokine transcription levels: In this experiment, the transcription levels of TNF-α, IL-6, IL8 and IL-10 genes in the small intestine of each group of mice were detected by RT-PCR. Fig.10 shown.

[0137] By detecting the pro-inflammatory cytokine TNF-α ( Fig.10 Middle (A) shows that compared with the ETEC group, the LngC group ( P <0.05), FaeC group ( P <0.05), MIX group ( P <0.001) and TNF-α levels were significantly downregulated, while there was no significant difference among the LngC group, FaeC group, and MIX group ( P >0.05). By detecting the pro-inflammatory factor IL-6 ( Fig.10 Middle (B) shows that compared with the ETEC group, the LngC group ( P <0.01), FaeC group ( P <0.001), MIX group ( P <0.0001) and IL-6 levels were significantly downregulated. In addition, the IL-6 level in the MIX group was significantly lower than that in the LngC group ( P <0.05), FaeC group ( P <0.01). By detecting the anti-inflammatory factor IL-10 ( Fig.10 Middle (C) shows that compared with ETEC, LngC group ( P <0.05), FaeC group ( P <0.001), MIX group ( P <0.0001) and IL-10 levels were significantly upregulated. Meanwhile, the level of anti-inflammatory factor IL-10 in the MIX group was significantly higher than that in the LngC group ( P <0.001), FaeC group ( P <0.05). By detecting the pro-inflammatory factor IL-8 ( Fig.10 The results in (D) showed that the FaeC group showed significant differences compared with the ETEC group ( P <0.01, and there was no statistical difference in other groups ( P >0.05).

[0138] In this experiment, RT-PCR was used to analyze the gene expression of key cytokines in the intestine of immunized piglets. The results showed that the expression of TNF-α and IL-6 in the MIX group was significantly lower than that in the ETEC group. In terms of IL-10 expression level, each immune group was significantly higher than the ETEC group, indicating that the immune response was well regulated. According to the above results, it can be judged that the large-scale release of intestinal proinflammatory factors caused by ETEC infection is closely related to the inhibition of anti-inflammatory factors. The use of engineered bacteria for prevention can help reduce the secretion of these proinflammatory factors and increase the level of anti-inflammatory factors, thereby reducing intestinal inflammation in piglets.

[0139] 2.3.4 Detection of inflammatory cytokine protein levels in piglets: The levels of TNF-α, IL-6 and IL-1β cytokines in the serum of each group of piglets were detected by ELISA. The test results are shown in Fig.11 ELISA detection of TNF-α ( Fig.11 (A) shows that, compared with the ETEC group, the LngC group ( P <0.0001) and FaeC group ( P <0.0001) in terms of TNF-α expression level, which was significantly higher than that of the ETEC group, followed by the PPG group ( P <0.01) and MIX group ( P <0.01) showed significant difference. ELISA detection of IL-6 ( Fig.11 Middle (B) shows that by comparing with the ETEC group, the LngC group showed a very significant difference ( P <0.001, followed by the FaeC group ( P <0.01), there was no statistical difference between the PPG group and the MIX group. ELISA detection of IL-1β ( Fig.11 Middle (C) shows that there is no significant difference between the groups compared with the ETEC group ( P >0.05). These experimental results show that the inflammatory factor protein content in the serum of piglets treated with different engineered bacteria is statistically significantly different. There are significant differences in inflammatory factors between the PBS group and the ETEC group, which can be used to determine that the experimental model is established. The downregulation trend of pro-inflammatory cytokines in the LngC group and the FaeC group is more obvious, and the MIX group does not show significant statistical differences in inflammatory cytokines.

[0140] 2.3.5 Effect of engineered bacteria on the expression of secretory IgA in piglets: In this experiment, the ELISA method was used to detect the content of sIgA in the intestines of challenged piglets. Fig.11As shown in (D). In the fecal sIgA test, the fecal sIgA level in the MIX group was significantly different from that in the ETEC group (p<0.0001), and the FaeC group was significantly different from that in the ETEC group (p<0.05). The sIgA level in the MIX group was significantly upregulated compared with the FaeC group (p<0.05).

[0141] 2.3.6 Results of histopathological observation of piglets: Morphological analysis of small intestine Fig.12 As shown in Figure 2. After ETEC infection, the intestinal villi of each group were damaged to varying degrees. Fig.12 Middle (D)), FaeC group ( Fig.12 The degree of intestinal villi damage in the middle (E) group was relatively mild, followed by the PPG group ( Fig.12 Middle (F))、MIX group( Fig.12 Middle (C)), ETEC challenge group ( Fig.12 The middle (B) group showed relatively severe damage, with sparse intestinal villi, different lengths, and structural changes, which was different from the PBS group ( Fig.12 The above research results show that immunization with Lactobacillus plantarum containing LngC and FaeC engineered strains can effectively reduce the intestinal villi damage caused by ETEC infection in piglets, thereby improving the structure of the intestinal mucosal barrier.

[0142] The results of this experiment showed that NC8-pPG612.1-FaeC and NC8-pPG612.1-LngC can activate the intestinal mucosal immune response, reduce the intestinal damage caused by ETEC, protect the intestinal villus structure, and improve the body's ability to resist ETEC infection. The mixed engineered bacteria immunized piglets in terms of immune cells and inflammatory factors, which was better than the immune effect of a single engineered bacteria. These results show that the use of recombinant Lactobacillus plantarum as a vaccine vector can not only directly combat the intestinal damage caused by ETEC, but also regulate the intestinal microenvironment, stimulate the body to produce a systemic immune response, reduce the morbidity and mortality of ETEC infection, and thus improve the economic benefits of the pig industry.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still make various modifications to the technical solution or replace some technical features with equivalents after reading this description. Any modification, equivalent replacement or improvement made within the spirit and principle of the present invention shall be deemed to fall within the protection scope of the present invention.

Claims

1. A novel functional lactic acid bacterium expressing ETEC antigen, characterized in that: The novel functional lactic acid bacteria expressing ETEC antigens uses Lactobacillus plantarum NC8 as a host bacterium and pPG612.1 as an expression vector, wherein the pPG612.1 expression vector contains the LngC gene of ETEC; the novel functional lactic acid bacteria expressing ETEC antigens expresses and produces LngC protein; Wherein, the nucleotide sequence of the LngC gene is shown in SEQ ID NO:

2.

2. A composition comprising the novel functional lactic acid bacteria expressing ETEC antigens according to claim 1.

3. The composition according to claim 2, characterized in that The composition further comprises the following recombinant Lactobacillus plantarum: the recombinant Lactobacillus plantarum uses Lactobacillus plantarum NC8 as a host bacterium and pPG612.1 as an expression vector, and the pPG612.1 expression vector contains the FaeC gene of ETEC; The recombinant Lactobacillus plantarum expresses and produces FaeC protein, and the nucleotide sequence of the FaeC gene is shown in SEQ ID NO:

1.

4. A product comprising the novel functional lactic acid bacteria expressing ETEC antigens according to claim 1 or the composition according to claim 2 or 3.

5. The product according to claim 4, characterized in that The product is a microecological preparation, a pharmaceutical preparation or a feed additive.

6. The product according to claim 5, characterized in that The pharmaceutical preparation is an oral live vector vaccine.

7. A method for constructing the new functional lactic acid bacteria expressing ETEC antigens according to claim 1, characterized in that: include: Obtain the LngC gene fragment of ETEC; The gene fragment was constructed into the pPG612.1 expression vector; The pPG612.1 expression vector containing the gene fragment was introduced into Lactobacillus plantarum NC8.

8. Use of the novel functional lactic acid bacteria expressing ETEC antigens according to claim 1 or the composition according to claim 2 or 3 in the preparation of a product having at least one of the following uses: (1) Products for preventing or treating ETEC infection; (2) Immunomodulatory products; (3) Anti-inflammatory products; (4) Intestinal protection products; in, The product is a microecological preparation, a pharmaceutical preparation or a feed additive.

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