Anti-ETEC engineering probiotics as well as construction method and application thereof

Abstract

The invention provides an anti-ETEC engineering probiotic as well as a construction method and application thereof, and belongs to the technical field of gene engineering. According to the present invention, lactic acid bacteria are adopted as a presentation vector, a CLA gene coding linoleic acid isomerase in Lactobacillus rhamnosus GG (Lactobacillus rhamnosus) is amplified, and the CLA gene is connected with a Lactobacillus casei ATCC393 (Lactobacillus casei) expression vector pPG 612 so as to construct the engineering bacteria probiotic LC-pPG-CLA; the engineering probiotics have high adhesion and invasion ability to Caco-2 cells, and can competitively inhibit the ETEC K88 strain from adhering to the Caco-2 cells so as to reduce invasion and infection of the ETEC K88 strain to the Caco-2 cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to an anti-ETEC engineered probiotic, a construction method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Enterotoxigenic Escherichia coli (Enterotoxigenic Escherichia coli ETEC is one of the common human-animal shared infectious pathogenic bacteria, which can cause diarrhea in humans and animals. Porcine pathogenic infection caused by E. coli, also known as porcine colibacillosis, can cause a series of diseases such as neonatal diarrhea, post-weaning diarrhea, edema disease, septicemia, polyserositis, E. coli mastitis and urinary tract infection. Due to the increasing selective pressure of using antibiotics to treat E. coli infection, the emergence of antimicrobial resistance (AMR) limits the treatment options of breeders.

[0004] The classic model of ETEC pathogenesis requires two processes to initiate infection. The first process is the colonization of ETEC on the small intestinal epithelium through adhesins, which is necessary for ETEC to release enterotoxins; secondly, various toxins released into the host epithelial cells, mainly including heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). It is generally believed that LT and ST enterotoxins cause host diarrhea by increasing cyclic nucleotide synthesis, leading to electrolyte and water loss. Although existing vaccines can provide some preventive protection, some vaccines do not target the toxicity caused by the main ST enterotoxin, while others only target a single adhesin or enterotoxin. The immune effect of the vaccine is not ideal. Therefore, it is of great significance to develop a new broad-spectrum vaccine to prevent diarrhea in newborn piglets.

[0005] Lactic acid bacteria (LAB) is a very potential mucosal vaccine carrier. With the development of genetic engineering, laboratory strains mainly of cocci and bacilli have been used to express a series of heterologous antigens. A large number of studies have shown that LAB mucosal vaccines can stimulate all branches of the immune system and provide adequate protection against pathogen infection.

[0006] Conjugated linoleic acid (CLA) is a class of active metabolites that has a certain regulatory effect on the intestinal barrier; linoleate isomerase is a key enzyme for producing conjugated linoleic acid.

[0007] At present, the prior art provides K88ac +Construction method of attenuated enterotoxigenic Escherichia coli strain: adopt K88ac knockout + ST II gene of enterotoxigenic Escherichia coli, achieve K88ac attenuation + Enterotoxigenic Escherichia coli. However, the gene knockout technology is time-consuming and expensive, and may also have side effects.

[0008] In summary, in terms of resisting enterotoxigenic Escherichia coli infection, a safer and more effective solution is urgently needed. SUMMARY

[0009] In view of the deficiencies of the prior art, the present application provides an anti-ETEC engineering probiotic and a construction method and application thereof.

[0010] The first aspect of the present application is to provide an anti-ETEC engineering probiotic, which is a delivery carrier of lactic acid bacteria, and is constructed by amplifying Lactobacillus rhamnosus (LGG) Lactobacillus rhamnosus GG) encoding linoleic acid isomerase and connecting the CLA gene with the Lactobacillus expression vector pPG612, and the obtained engineering probiotic LC-pPG-CLA is constructed, and the CLA nucleotide sequence table is shown as SEQ ID NO. 3.

[0011] The second aspect of the present application is to provide a construction method of the engineering probiotic LC-pPG-CLA, comprising the following steps: The pPG-CLA plasmid with the nucleotide sequence shown as SEQ ID NO. 4 is electroporated into L.casei ATCC 393 competent cells, and the engineering probiotic LC-pPG-CLA is obtained after culture.

[0012] Further, the construction method of the pPG-CLA plasmid comprises the following steps: S1, amplification of the CLA gene; PCR amplification of the CLA gene encoding linoleic acid isomerase in Lactobacillus rhamnosus is carried out by taking the Lactobacillus rhamnosus genome as a template, and the target gene is recovered; S2, construction of the pEASY-CLA recombinant plasmid; The CLA target gene fragment recovered in the above step is connected with the cloning vector pEASY-Blunt-Zero, and the connection product is transformed into the MC1061 competent cells, and the pEASY-CLA recombinant plasmid is obtained after culture; S3, construction of the pPG-CLA recombinant plasmid, comprising the following steps: The Escherichia coli containing the pPG612 plasmid was activated and the plasmid was extracted; the expression vector pPG612 and the pEASY-CLA recombinant plasmid obtained in step S2 were double-digested respectively; after the digestion, agarose gel electrophoresis was performed to recover the target gene and vector fragment; the target gene and the expression vector pPG612 were ligated and transformed into MC1061 competent cells. After cultivation, the plasmid was extracted and double-digested and PCR identified. The suspected plasmid was sent for sequencing and named pPG-CLA after alignment.

[0013] In some embodiments, the double digestion enzymes are restriction endonucleases SmaI and NcoI.

[0014] In some embodiments, T4 DNA ligase is used to ligate the gene of interest and the expression vector pPG612.

[0015] The third aspect of the present invention is to provide the use of the anti-ETEC engineered probiotics in the preparation of anti-ETEC drugs.

[0016] Furthermore, the drug has at least one of the following effects: (1) Improve the rapid weight loss and diarrhea symptoms caused by ETEC K88 infection; (2) Inhibit colon shortening symptoms caused by ETEC K88 infection; (3) Upregulate the expression levels of anti-inflammatory factor IL-10 and intestinal tight junction proteins Occludin-1, ZO-1, and p65 genes, maintaining intestinal epithelial cell permeability and intestinal barrier function; (4) Alleviate inflammatory damage caused by ETEC; (5) Down-regulate the gene expression levels of pro-inflammatory factors IL-1β and TNF-α; (6) Improve intestinal villus damage; (7) Increase the abundance of Firmicutes and Bacteroidetes, and reduce the relative abundance of harmful bacteria Proteobacteria.

[0017] The fourth aspect of the present invention is to provide the use of the anti-ETEC engineered probiotics described in the first aspect in the preparation of anti-inflammatory bowel disease drugs, especially in the preparation of drugs for preventing and / or treating enterotoxigenic Escherichia coli infections.

[0018] The medicine is an oral preparation, including tablets, capsules, oral liquid or granules.

[0019] The fifth aspect of the present invention is to provide a vaccine comprising the anti-ETEC engineered probiotics described in the first aspect, for preventing Escherichia coli infection.

[0020] The beneficial effects achieved by one or more embodiments of the present invention are as follows: 1. The present invention successfully constructed an engineered bacterium LC-pPG-CLA, which has high adhesion and invasion capabilities on Caco-2 cells. It can competitively inhibit the adhesion of the ETEC K88 strain to Caco-2 cells, reducing the invasion and infection of the ETEC K88 strain on Caco-2 cells, thereby preventing and / or treating enterotoxigenic Escherichia coli infection.

[0021] 2. Oral administration of the engineered probiotic LC-pPG-CLA to mice can significantly increase the number of B cell proliferation in the spleen (SP), Peyer's patches (PPs) and mesenteric lymph nodes (MLN) of the experimental animals, promote B lymphocyte proliferation, activate dendritic cells, promote Treg cell differentiation and maturation, upregulate the expression levels of the anti-inflammatory factor IL-10 and intestinal tight junction proteins Occludin-1, ZO-1, and p65 genes, and downregulate the expression levels of the pro-inflammatory factors IL-1β and TNF-α genes.

[0022] 3. The engineered probiotic LC-pPG-CLA provided by the present invention can improve intestinal villus damage and effectively protect mice infected with ETECK88; through species composition analysis, it was found that the engineered probiotic LC-pPG-CLA can increase the abundance of Firmicutes and Bacteroidetes, and reduce the relative abundance of harmful bacteria Proteobacteria.

[0023] 4. The engineered probiotic LC-pPG-CLA prepared by this invention has excellent cell adhesion ability. Oral immunization of mice can promote humoral and cellular immune responses and effectively alleviate the inflammatory damage caused by ETEC. This is conducive to the development of new oral vaccines and lays a foundation for the research and development of oral formulations against ETEC infection. It is also of great significance for the development of new broad-spectrum vaccines to prevent diarrhea in newborn piglets. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings, which constitute 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 improper limitations on the present invention.

[0025] Figure 1 The figure shows the amplification results of the CLA gene of the recombinant LGG strain; lanes 1-2 show the PCR amplification results of the CLA gene.

[0026] Figure 2A is a PCR identification diagram of the recombinant plasmid pEASY-CLA, and lane 1 is the PCR amplification result of the pEASY-CLA recombinant plasmid; B is a PCR identification diagram of the recombinant plasmid pPG-CLA, and lane 1 is the PCR amplification result of the pEASY-CLA recombinant plasmid.

[0027] Figure 3 This is the map of the recombinant plasmid pPG-CLA.

[0028] Figure 4 The figure below shows the identification of recombinant lactic acid bacteria; A shows the PCR identification of recombinant lactic acid bacteria LC-pPG-CLA plasmid, and lane 1 shows the PCR amplification result of LC-pPG-CLA recombinant plasmid; B shows the Western blot identification of recombinant lactic acid bacteria LC-pPG-CLA, and lanes 1-2 show the immunoblotting detection results of recombinant protein of LC-pPG-CLA strain.

[0029] Figure 5 Indirect immunofluorescence identification of recombinant lactic acid bacteria LC-pPG-CLA; A is the control group (white light, plasmid-free strain), and B is the LC-pPG-CLA recombinant strain (green fluorescence).

[0030] Figure 6 A is the result of the ability test of the engineered bacteria; A is the result of the adhesion ability test of the engineered bacteria; B is the result of the competitive adhesion ability test of the engineered strain; C is the result of the invasion ability test of the engineered bacteria; D is the result of the competitive invasion ability test of the engineered bacteria.

[0031] Figure 7 Figure 3 is a graph showing weight changes in C57BL / 6 mice after infection; A is a C57BL / 6 mouse; B is a C57BL / 6 dirty cage mouse.

[0032] Figure 8 Figure 2 is a graph showing the colon length test results of C57BL / 6 mice after infection; Figure 3 is a graph showing the colon length test results of C57BL / 6 mice after infection; Figure 4 is a graph showing the colon length test results of C57BL / 6 mice in dirty cages.

[0033] Figure 9 B220 in SP, PPs, and MLN of C57BL / 6 mice + IgA + B lymphocyte proliferation chart; A is the statistical chart of the cell number detection results in SP; B is the statistical chart of the cell number detection results in PPs; C is the statistical chart of the cell number detection results in MLN.

[0034] Figure 10 CD11c in SP, PPs, and MLN of C57BL / 6 mice +Figure 4 shows the activation of DC dendritic cells; A is a statistical diagram of the cell number detection results in SP; B is a statistical diagram of the cell number detection results in PPs; C is a statistical diagram of the cell number detection results in MLN.

[0035] Figure 11 CD25 in SP, PPs, and MLN of C57BL / 6 mice + FOXP3 + Figure 3. T lymphocyte proliferation. A is a statistical graph showing the results of cell number detection in SP. B is a statistical graph showing the results of cell number detection in PPs. C is a statistical graph showing the results of cell number detection in MLN.

[0036] Figure 12 Figure 1 is the result of IL-10 content detection in the serum of C57BL / 6 mice; A is the IL-10 content in the serum of C57BL / 6 mice after the first immunization; B is the IL-10 content in the serum of C57BL / 6 mice after the second immunization; C is the IL-10 content in the serum of C57BL / 6 dirty cage mice.

[0037] Figure 13 The results of the IgG content test in the serum of C57BL / 6 mice are shown in Figure 2. A is the IgG content in the serum of C57BL / 6 mice after the first immunization; B is the IgG content in the serum of C57BL / 6 mice after the second immunization; and C is the IgG content in the serum of C57BL / 6 mice in dirty cages.

[0038] Figure 14 The results of the sIgA content test in the feces of C57BL / 6 mice; A is the sIgA content in the feces of C57BL / 6 mice after the first immunization; B is the sIgA content in the feces of C57BL / 6 mice after the second immunization; C is the sIgA content in the feces of C57BL / 6 mice in dirty cages.

[0039] Figure 15 Figure 2 is the result of IL-1β content detection in the serum of C57BL / 6 mice; A is the IL-1β content in the serum of C57BL / 6 mice after the first immunization; B is the IL-1β content in the serum of C57BL / 6 mice after the second immunization; C is the IL-1β content in the serum of C57BL / 6 dirty cage mice.

[0040] Figure 16 Figure 2 is a graph showing the results of TNF-α content detection in the serum of C57BL / 6 mice; A is the TNF-α content in the serum of C57BL / 6 mice after the first immunization; B is the TNF-α content in the serum of C57BL / 6 mice after the second immunization; C is the TNF-α content in the serum of C57BL / 6 mice in dirty cages.

[0041] Figure 17These are the results of the detection of Occludin-1 and ZO-1 gene expression levels in the small intestine of C57BL / 6 mice; A is the Occludin-1 gene expression level in C57BL / 6 mice; B is the ZO-1 gene expression level in C57BL / 6 mice; C is the p65 gene expression level in C57BL / 6 mice.

[0042] Figure 18 These are the results of detecting the expression levels of IL-1β, TNF-α, and IL-10 genes in the small intestine of C57BL / 6 mice; A is the expression level of IL-1β gene in C57BL / 6 mice; B is the expression level of TNF-α gene in C57BL / 6 mice; C is the expression level of IL-10 gene in C57BL / 6 mice.

[0043] Figure 19 This is a phylum-level analysis of the intestinal microbial composition of C57BL / 6 mice.

[0044] Figure 20 These are the results of intestinal histopathological examination of C57BL / 6 mice after infection; A is a C57BL / 6 mouse; B is a C57BL / 6 dirty cage mouse. DETAILED DESCRIPTION

[0045] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0046] In some embodiments of the present invention, an anti-ETEC engineered probiotic is provided, wherein the anti-ETEC engineered probiotic is amplified by using lactic acid bacteria as a presentation vector and Lactobacillus rhamnosus ( Lactobacillus rhamnosus The CLA gene encoding linoleic acid isomerase in GG was ligated with the Lactobacillus expression vector pPG612 to construct the engineered probiotic LC-pPG-CLA.

[0047] In some embodiments of the present invention, a method for constructing the engineered probiotic LC-pPG-CLA is also provided, comprising the following steps: The pPG-CLA plasmid with the nucleotide sequence shown in SEQ ID NO.4 was electroporated into L.casei The engineered probiotic LC-pPG-CLA was obtained after culture in ATCC 393 competent state.

[0048] The construction method of the pPG-CLA plasmid comprises the following steps: S1, CLA gene amplification: Using the Lactobacillus rhamnosus genome as a template, PCR amplify the CLA gene encoding linoleic acid isomerase in Lactobacillus rhamnosus; and recover the target gene; S2, construction of pEASY-CLA recombinant plasmid: ligate the CLA target gene fragment recovered in the previous step with the cloning vector pEASY-Blunt-Zero; take the ligation product and transform it into MC1061 competent cells using conventional chemical transformation methods, and culture it to obtain the pEASY-CLA recombinant plasmid; S3, construction of pPG-CLA recombinant plasmid: Escherichia coli containing pPG612 plasmid was activated and the plasmid was extracted; the recombinant plasmid pEASY-CLA and the expression vector pPG612 were double-digested respectively; after the digestion, the target gene and vector fragments were detected by agarose gel electrophoresis, and the target gene and expression vector fragments were recovered by gel electrophoresis; the target gene and expression vector pPG612 were ligated and transformed into MC1061 competent cells. After culture, the plasmid was extracted and double-digested and PCR identified. The suspected plasmid was sent for sequencing and named pPG-CLA after alignment.

[0049] In some embodiments, the double enzyme digestion enzyme is a restriction endonuclease Sma I. Nco I.

[0050] In some embodiments, T4 DNA ligase is used to ligate the gene of interest and the expression vector pPG612.

[0051] In some embodiments of the present invention, use of the engineered probiotic LC-pPG-CLA in the preparation of anti-ETEC drugs is provided.

[0052] Furthermore, the drug has at least one of the following effects: (1) Improve the rapid weight loss and diarrhea symptoms caused by ETEC K88 infection; (2) Inhibit colon shortening symptoms caused by ETEC K88 infection; (3) Upregulate the expression levels of anti-inflammatory factor IL-10 and intestinal tight junction proteins Occludin-1, ZO-1, and p65 genes, maintaining intestinal epithelial cell permeability and intestinal barrier function; (4) Alleviate inflammatory damage caused by ETEC; (5) Down-regulate the gene expression levels of pro-inflammatory factors IL-1β and TNF-α; (6) Improve intestinal villus damage; (7) Increase the abundance of Firmicutes and Bacteroidetes, and reduce the relative abundance of harmful bacteria Proteobacteria.

[0053] In some embodiments of the present invention, there is provided the use of the engineered probiotic LC-pPG-CLA in the preparation of anti-inflammatory bowel disease drugs, especially in the preparation of drugs for preventing and / or treating enterotoxigenic Escherichia coli infections.

[0054] The medicine is an oral preparation, including tablets, capsules, oral liquid or granules.

[0055] The medicine is a vaccine for preventing Escherichia coli infection.

[0056] In some embodiments of the present invention, a vaccine is provided, comprising the anti-ETEC engineered probiotic bacteria, for preventing Escherichia coli infection.

[0057] Sources of the biological materials (genes, vectors, microorganisms, etc.) involved in the present invention: 1. Escherichia coli MC1061, Lactobacillus casei ATCC393, and vector pPG612 were purchased from BNCC. 2. pEASY-Blunt-Zero cloning vector kit was purchased from Quanshijin (Beijing) Biological Co., Ltd.; 3. ETEC K88 strain and human colorectal cancer epithelial cell line Caco-2 were purchased from BNCC.

[0058] The present invention will be further described below with reference to the embodiments.

[0059] Example 1, Construction and Verification of Engineered Probiotic LC-pPG-CLA 1. CLA gene amplification and cloning vector construction 1.1 Primer design for the CLA gene The complete genomic data of LGG were obtained from the NCBI database, with the genome GenBank number: NC_013198.1 and the CLA gene sequence information, with the GeneBank number: CP031290.1. A pair of specific upstream and downstream primers with enzyme cleavage sites were designed based on the pPG612 expression vector sequence. The specific primer sequence information is as follows, and the underline represents the enzyme cleavage site.

[0060] F: 5'- CCATGG ATGCACCATCATCATCATCATTTCAAACATTTTCTTGGTTGCGCGC-3'( Nco I) (as shown in the sequence listing SEQ ID NO.1); R: 5'- CCCGGG ATGATGATGATGATGGTGCATATGATCAAGCATAAGGCGATCATGA-3'( Sma I) (as shown in the sequence listing SEQ ID NO.2).

[0061] Primers were synthesized by Changchun Kumei Biotechnology Co., Ltd. and the genome of Lactobacillus rhamnosus GG was used as a template. PCR amplification was performed using the upstream and downstream primers. The expected size of the CLA gene fragment was 1692 bp. The amplification system is shown in Table 1. The amplification results of the LGG genome and the amplification of the CLA gene are shown in Figure 1. Figure 1 shown.

[0062] Table 1 PCR amplification reaction system

[0063] PCR amplification reaction conditions: pre-denaturation-denaturation-annealing-extension-re-extension, 30 cycles at 94°C for 5 min, 94°C for 1 min, 67°C for 1 min, and 72°C for 10 min. After completion of the reaction, 5 μL of the final PCR product was analyzed by agarose gel electrophoresis and visualized using a UV gel imaging system. The target gene was recovered and verified to be of the expected size according to the gel recovery kit instructions and stored at -20°C.

[0064] 1.2 Construction and identification of pEASY-CLA recombinant plasmid The CLA gene fragment recovered from the gel in the previous step was ligated with the cloning vector pEASY-Blunt-Zero at 16°C for 12 h. The ligation system is shown in Table 2.

[0065] Table 2 Ligation reaction system

[0066] Take 5 μL of the ligation product and transform it into the MC1061 competent medium using ordinary chemical transformation. The specific steps are: take out the MC1061 competent medium at -80℃, ice bath for 15 min, transfer the ligation product to the competent medium, mix well, ice bath for 30 min, heat shock at 42℃ for 90 s, add 800 μL LB broth, shake on a 37℃ constant temperature shaker for 2 h, centrifuge at 5000 r / min for 10 min, discard the supernatant, keep 50 μL of the supernatant, blow off the bacterial precipitation, evenly spread LB agar (containing 50 μg / mL kanamycin), culture in a 37℃ incubator for 12 h, pick a single colony, extract the plasmid according to the instructions of the bacterial plasmid extraction kit for PCR identification, send the suspected plasmid to Changchun Kumei Biological Company for sequencing, and after identification, it is named pEASY-CLA. The PCR identification figure of the recombinant plasmid pEASY-CLA is shown as follows Figure 2 As shown in A.

[0067] 1.3 Construction and identification of pPG-CLA recombinant plasmid 1.3.1 Preparation of MC1061 competent cells Take out the laboratory stored in -80℃ freezer E.coliMC1061 was activated and streaked onto LB agar after 2-3 generations. A single colony was picked and placed in LB broth and cultured at 37°C with shaking for 12 h. The next day, the bacterial suspension was inoculated into 100 mL LB broth at a ratio of 1.5% and cultured at 37°C with shaking until OD 600 = about 0.5, transfer the cultured bacterial solution into a 50 mL centrifuge tube, place on ice for 10 min, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, gently suspend the bacteria with 10 mL pre-cooled 0.1M CaCl2, place on ice for 10 min, centrifuge at 4°C, 5000 rpm for 10 min, discard the supernatant, repeat washing twice, let it stand on ice for 20 min, finally gently suspend the bacteria with 2 mL pre-cooled 0.1M CaCl2 containing 25% glycerol, ice bath for 30 min, and dispense the prepared competent cells into pre-cooled centrifuge tubes at 50 uL / tube and store at -80°C for later use.

[0068] 1.3.2 Ligation and transformation of pPG-CLA recombinant plasmid E. coli carrying the pPG612 plasmid was activated and streaked onto LB agar (containing 10 μg / mL chloramphenicol) after 2-3 generations. A single colony was picked and placed in LB broth and cultured on a shaker at 37°C for 12 h to extract the plasmid. Restriction enzymes were used to Sma I. Nco I. Double enzyme digestion was performed on the recombinant plasmid pEASY-CLA and the expression vector pPG612, respectively. The double enzyme digestion system is shown in Table 3. The digestion reaction was carried out at 37°C for 4 h. After the digestion, the target gene and vector fragments were recovered by agarose gel electrophoresis.

[0069] Table 3 Double enzyme digestion reaction system

[0070] The CLA gene was cloned into the expression vector pPG612 using T4 DNA ligase, ligated at 16°C for 12 h, and transformed into MC1061 competent cells the next day. The ligation system is shown in Table 4. The bacterial culture solution, which had been cultured for 2 h, was centrifuged and evenly spread on LB agar (containing 10 μg / mL chloramphenicol). The cells were cultured at 37°C overnight, and single colonies were picked. The plasmids were extracted and subjected to double enzyme digestion and PCR identification. Suspected plasmids were sent for sequencing. The recombinant plasmid was named pPG-CLA (as shown in the sequence listing, SEQ ID NO. 4). The PCR identification of the recombinant plasmid pPG-CLA is shown in Figure 4. Figure 2 As shown in B, the structure is as follows Figure 3 shown.

[0071] Table 4 Ligation reaction system

[0072] 1.4 Construction and genetic verification of the LC-pPG-CLA recombinant strain 1.4.1 L.casei Preparation of ATCC393 competent cells Pick L.casei The ATCC393 strain was activated and streaked onto MRS agar without antibiotics. The culture was anaerobically cultured at 37°C for 12 h. A single colony was picked and cultured. The next day, the bacterial solution was inoculated into 200 mL MRS broth at a ratio of 2% and cultured until the OD 600 =0.6-0.8. Transfer the bacterial suspension to a centrifuge tube, place on ice for 30 min, and centrifuge at 5000 rpm for 10 min at 4°C. Collect the bacterial pellet, wash twice with 20 mL of pre-chilled EPWB, then once with 20 mL of pre-chilled EPB, and finally resuspend in 1 mL of pre-chilled EPB. Place on ice for 15 min, and store at 100 μL per tube in a -80°C refrigerator.

[0073] 1.4.2 Electrotransformation of recombinant plasmids in lactic acid bacteria The correctly sequenced pPG-CLA plasmid was electroporated into L.casei In the ATCC393 competent cell, the specific steps are as follows: thaw the competent cell on ice, take 10 μL of the recombinant plasmid pPG-CLA and mix it with the competent cell, ice bath for 5 minutes, transfer it to a pre-cooled 2 mm electric shock cup, and use an electric rotator to shock (parameters 2.68 KV, 3 ms). After the end, immediately add 800 μL of recovery medium (MRS broth containing 15% sucrose), ice bath for 15 minutes, and incubate anaerobically at 37°C for 3 hours. Take 100 μL of the turbid bacterial liquid to spread on MRS agar plate (containing 10 μg / mL chloramphenicol), and incubate anaerobically at 37°C for 24 hours.

[0074] The next day, single colonies were picked and incubated in MRS broth (containing 10 μg / mL chloramphenicol) at 37°C for 12 h. Four mL of the bacterial suspension was used for Gram-positive bacterial plasmid extraction. 250 μL of lysozyme (100 mg / mL) was added for bacterial cell disruption for 1 h. The remaining steps were performed according to the instructions of the Kangwei Century Bacterial Plasmid Extraction Kit. Suspected plasmids were identified by PCR and double enzyme digestion, and the suspected plasmids were sent for sequencing. The strain with the correct sequence was named LC-pPG-CLA.

[0075] 1.5 Western blot identification of recombinant lactic acid bacteria 1.5.1 Preparation of recombinant lactic acid bacteria protein The recombinant lactic acid bacteria LC-pPG-CLA was inoculated into MRS broth (containing 10 μg / mL chloramphenicol) for culture. The next day, the bacterial solution was inoculated into 2% lactose MRS broth (containing 10 μg / mL chloramphenicol) at a ratio of 4% and anaerobically induced at 37°C for 12 hours. Uninduced bacteria served as a negative control. 10 mL of the induced and uninduced bacterial solutions were collected by centrifugation at 8000 rpm for 15 minutes at 4°C to collect the bacterial pellets. The pellets were washed three times with sterile PBS and resuspended in 500 μL of lysozyme. The pellets were broken at 37°C for 1 hour. The cells were collected by centrifugation under the same conditions, resuspended in 200 μL of PBS, and mixed with 50 μL of 5× SDS PAGE Buffer. The mixture was boiled at 100°C for 10 minutes and stored at -80°C. The PCR identification results of the recombinant lactic acid bacteria LC-pPG-CLA are shown in Figure 2. Figure 4 As shown in A.

[0076] 1.5.2 SDS-PAGE Refer to the instructions of the YAZYME PAGE Gel Rapid Preparation Kit to prepare stacking gel and separation gel, where the gel spacing is 1.5 mm. The specific steps are as follows: (1) Take equal volumes of lower gel solution and 4.0 mL of lower gel buffer and mix well; (2) Add 80 μL of the modified coagulant to the mixed solution in step 1 and mix well; (3) Pour the mixed solution from step 2 into the glass plate and add appropriate amount of water or alcohol to seal it; (4) After the lower layer of gel solidifies, pour off the upper layer of water or alcohol; (5) Take equal volumes of upper gel solution and 1.0 mL of colored upper gel buffer and mix well; (6) Add 20 μL of the modified coagulant to the mixed solution in step 5 and mix well; (7) Pour the mixed solution from step 6 into the glass plate and insert the comb teeth; (8) After the upper layer of gel solidifies, remove the comb teeth and use it for electrophoresis.

[0077] (9) Add 1 L of 1× SDS-PAGE electrophoresis buffer to the electrophoresis tank, load 5 μL of rainbow protein marker and 10 μL of protein, and perform electrophoresis at a constant voltage of 80 V for 120 min.

[0078] 1.5.3 Western Blot (1) After SDS-PAGE electrophoresis, discard the stacking gel and retain the separation gel. Soak the PVDF membrane in methanol solution in advance for activation (about 1 min), and soak the filter paper, gel, sponge, etc. in the transfer solution; (2) Transfer: Place the membrane in the order from negative electrode to positive electrode: sponge - three-layer filter paper - PVDF membrane - glue - three-layer filter paper - sponge, add transfer solution, place on ice, and transfer at a constant current of 300 mA for 90 min; (3) Blocking: Take out the PVDF membrane and place it in blocking solution for 2 h at room temperature; (4) After blocking, wash the membrane three times with TBST, 5 min each time; (5) Incubation with primary antibody: Add anti-His-Tag primary antibody (antibody diluted 1:3000 in TBST) and incubate at 4°C for 12 h; (6) Incubation with secondary antibody: Recover the primary antibody, wash the membrane three times with TBST, 5 min each time, add HRP-labeled goat anti-rabbit IgG secondary antibody (TBST 1:3000 diluted antibody 1:3000), and incubate on a shaker at room temperature for 2 h; (7) Exposure and development: Wash the membrane with TBST as in step 4, add ECL luminescent solution and place it in the imaging system for development, and record the results. Western blot identification of recombinant lactic acid bacteria LC-pPG-CLA is as follows Figure 4 As shown in B.

[0079] 1.6 Indirect immunofluorescence detection of recombinant lactic acid bacteria After the recombinant lactic acid bacteria LC-pPG-CLA was induced with lactose for 12 hours, 500 μL of the induced bacterial solution was taken and centrifuged at 8000 rpm for 10 minutes. The cells were washed three times with sterile PBS, and 200 μL of His-Tag was added. The cells were incubated at 37°C for 4 hours. The cells were washed three times with PBS and centrifuged under the same conditions. 200 μL of FITC-labeled IgG was added. The cells were incubated at 37°C in the dark for 4 hours. The cells were fixed with paraformaldehyde and observed inverted under a fluorescence microscope. The results of indirect immunofluorescence identification of recombinant lactic acid bacteria LC-pPG-CLA are shown in Figure 2. Figure 5 As shown in A and B: The LC-pPG-CLA recombinant strain can produce specific green fluorescence, while the control strain without plasmid has no specific green fluorescence, indicating that pPG-CLA can be expressed normally.

[0080] Example 2, Analysis of Biological Characteristics of Engineered Probiotics 1. Detection of Adhesion Ability of Engineered Bacteria Caco-2 cells (1 × 10 5 The cells were inoculated into 24-well plates and cultured until a monolayer of cells was formed. The confluent monolayer of cells was washed twice with pre-warmed PBS and the cells and bacteria (1 × 10 5 cells and 1×10 7CFU / mL of bacteria were used for the experiment), and the cells were co-cultured at 37°C for 3 h. The bacterial solution was discarded and washed three times with PBS to thoroughly remove unadhered bacteria. The entire process was performed gently to prevent cell detachment. 1% Triton X-100 was added for 30 min to lyse the cells. After serial dilution, the cells were plated on MRS agar. The number of adherent cells and bacteria was calculated by counting the colonies. Three replicates were performed per group to reduce experimental error. Adhesion rate (%) = colony count after adhesion / colony count before adhesion × 100%. The results are shown in Figure 2. Figure 6 As shown in A, the constructed recombinant bacteria LC-pPG-CLA can adhere to intestinal epithelial cells. The adhesion rate of LC-pPG-CLA cells is about 9.3%, which is significantly higher than that of other groups ( P <0.05).

[0081] 2. Competitive Adhesion Test of Engineered Probiotics to ETEC K88 Will L.casei The concentrations of ATCC 393 (labeled as LC in the figure), LC-pPG-CLA (labeled as LC-pPG-CLA in the figure), and ETECK88 (labeled as K88 in the figure) were adjusted to 1×10 7 CFU / mL, then K88 was mixed with the two strains in a 1:1 ratio. The control group (marked as Control in the figure) used an equal volume of PBS buffer instead of the two strains and mixed with K88 in a 1:1 ratio. They were inoculated into 24-well plates and incubated with Caco-2 cells at 37°C 5% CO2 for 3 hours. The culture medium was discarded, and 1% TritonX-100 was added for 30 minutes to lyse the cells. After gradient dilution, the cells were spread on MRS agar. The colony count was the number of bacteria adhering to the cells. Three replicates were performed in each group to reduce experimental errors. The results are shown in Figure 2. Figure 6 As shown in B, the cell adhesion of the engineered bacteria LC-pPG-CLA was significantly higher than that of the other groups ( P <0.05).

[0082] 3. Engineering Bacteria Invasion Test Use incomplete culture medium to adjust the concentration of engineered bacteria to 1×10 7 CFU / mL, Caco-2 cells (1×10 5 The cells were cultured to a monolayer, treated with bacteria and cells at an MOI of 1:100, incubated at 37°C for 3 h, washed three times with PBS to remove bacteria not adhering to the cell surface, and treated with MEM medium (containing 100 mg / mL gentamicin) for 2 h to remove bacteria not adhering to the cell surface. Finally, the cells were lysed with 1% TritonX-100 for 30 min, and the cells were spread on MRS agar after gradient dilution and incubated at 37°C for 12 h. The number of bacteria invading the cells was counted, and three replicates were used for each group. The results are shown in Figure 2. Figure 6As shown in C, the invasion ability of the engineered bacteria LC-pPG-CLA was significantly higher than that of other groups ( P <0.05).

[0083] 4. Competitive Invasion Assay of Engineered Probiotics The same co-culture step as in step 2 was performed. After discarding the culture medium, 200 μL of MEM medium (containing 100 mg / mL gentamicin) was added and cultured at 37°C for 2 h. The cells were lysed with 1% TritonX-100 for 30 min, and the cells were spread on MRS agar after serial dilution. The cells were cultured at 37°C for 12 h. The number of colonies was calculated as the number of bacteria invading the cells. Three replicates were performed for each group. Figure 6 As shown in D, the competitive invasion ability of the engineered bacteria LC-pPG-CLA was significantly higher than that of other groups ( P <0.05).

[0084] Example 3: Evaluation of the immune effect of engineered probiotics 1. Engineered Probiotic Immunization Program C57BL / 6 mice were randomly divided into the following five groups: a. control group (PBS buffer), b. LC group, c. CLA group, dD-LC (LC dirty cage sharing group), and eD-CLA group (CLA dirty cage sharing group), with 12 mice in each group. The mice in the control group were orally gavaged with 200 μL PBS, and the mice in the LC group were orally gavaged with 200 μL 1×10 8 CFU / mL L.casei ATCC393, CLA group was gavaged with 200 μL 1×10 8 CFU / mL of LC-pPG-CLA were gavaged continuously for 5 days, and booster immunization was performed after one week. The mice in the D-LC group were housed in the same cage with the LC group without gavage, while the mice in the D-CLA group were housed in the same cage with the CLA group.

[0085] Immunization protocol: After 7 days of acclimation, mice were immunized by oral gavage according to Table 5. Fast for 2-4 hours before immunization and return to normal diet 1 hour after immunization. Immunizations were administered starting on day 1 of the experiment and continued for 5 consecutive days, with a booster immunization administered one week later. C57BL / 6 mice in group de received the same cages and feces as mice in groups ac. Bedding was changed every three days for all groups, and the mice's condition was observed daily and supplemented with appropriate food.

[0086] Table 5 Experimental animal groups and immunization procedures

[0087] 2. Changes in mouse body weight and colon after challenge One week after the last immunization, on the 25th day of the experiment, ETEC K88 (1×10 8 CFU / mL, 200 μL) were orally inoculated into C57BL / 6 mice, and the weight changes and colon length of each group of experimental animals were observed and recorded within 72 hours. Figure 7 A and B and Figure 8 As shown in Figures A and B, immunization of C57BL / 6 mice and C57BL / 6 dirty cage mice with the engineered probiotic LC-pPG-CLA can improve the rapid weight loss and diarrhea symptoms caused by ETEC K88 infection, maintain the animal weight at a stable state, and significantly inhibit the colon shortening symptoms caused by ETEC K88 infection.

[0088] 3. Detection of Immune-Related Cell Proliferation and Differentiation Mouse spleen (SP), mesenteric lymph nodes (MLNs), and Peyer's patches (PPs) were sterilely collected to prepare single-cell suspensions, which were then incubated with CD11c-percp-cy5.5, CD80-PE, and CD86-FITC antibodies at 4°C in the dark for 30 min for detection of CD11c. + Dendritic cell activation; add CD45R / B220-APC and IgA-FITC antibody and incubate at 4°C in the dark for 30 min to detect IgA + B lymphocyte proliferation; CD3-APC-Cy7, CD4-APC and CD8-PE antibodies were added and incubated at 4°C in the dark for 30 minutes to detect T lymphocyte proliferation. Figure 9 Middle A~C, Figure 10 Middle A~C, Figure 11 As shown in A to C, IgA in PPs of C57BL / 6 mice in the LC-pPG-CLA group + The number of B lymphocytes was significantly higher than that of the control group ( P <0.05); CD11c in SP, PPs and MLN of C57BL / 6 mice in LC-pPG-CLA group + The number of DCs cells was significantly higher than that of the control group ( P <0.05); FOXP3 expression in SP and PPs of C57BL / 6 mice in the LC-pPG-CLA group + The number of Treg cells was significantly higher than that of the control group ( P <0.05), indicating that the engineered probiotic LC-pPG-CLA can promote the proliferation of B cells, dendritic cells and T lymphocytes in the lymphoid tissues of C57BL / 6 mice. 4. Cytokine and Immunoglobulin Detection The ELISA method was used to detect the levels of IgG, IL-1β, IL-10, and TNF-α in the serum of immunized mice and the levels of sIgA in the fecal supernatant. Figure 12 Middle A~C, Figure 13 Middle A~C, Figure 14 Middle A~C, Figure 15 Middle A~C, Figure 16 As shown in Figures A to C, the engineered probiotic LC-pPG-CLA can significantly upregulate the level of the anti-inflammatory factor IL-10 and significantly inhibit the levels of the pro-inflammatory factors IL-1β and TNF-α in the serum of experimental animals. In addition, the engineered probiotic LC-pPG-CLA can significantly increase the levels of sIgA and IgG in the serum of immune mice and the content of sIgA in the feces.

[0089] Total RNA was extracted from the small intestine of C57BL / 6 mice and reverse transcribed into cDNA. The expression levels of Occludin-1, ZO-1, and p65 genes were detected by RT-PCR. Figure 17 As shown in A to C, the engineered probiotic LC-pPG-CLA can increase the expression levels of intestinal tight junction proteins ZO-1, Occludin-1 and p65, and maintain intestinal epithelial cell permeability and intestinal barrier function.

[0090] Total RNA was extracted from the small intestine of C57BL / 6 mice and reverse transcribed into cDNA. The expression levels of IL-1β, TNF-α, and IL-10 genes were detected by RT-PCR. Figure 18 As shown in Figures A to C, the engineered probiotic LC-pPG-CLA can increase the expression level of intestinal IL-10 gene, while inhibiting the expression levels of pro-inflammatory factors IL-1β and TNF-α genes, thereby enhancing the immune response of mice.

[0091] 5. Histopathological Examination After infection, the duodenum, jejunum, ileum, and colon tissues of C57BL / 6 mice were obtained and fixed by immersing them in 4% paraformaldehyde solution. The intestinal segments were generally fixed for about 24 hours. The fixed tissues were trimmed and placed in an embedding box. After gradient dehydration with alcohol and transparentization with xylene, they were placed in a 58°C oven and waxed. The wax immersion time for wax I, wax II, and wax III was 30 minutes each. After the wax immersion, they were quickly permeabilized and embedded. They were immediately permeabilized and embedded in an embedding machine. The slices were sliced ​​to a thickness of 4 μm and spread in a 42°C water bath. The slices were placed in an 80°C dry heat box for 1 hour. The dried slices were immersed in xylene I and xylene II for 10 minutes each. The slices were dehydrated in different concentrations of alcohol for 1 minute each and washed twice with water; hematoxylin stained for 5 minutes and washed twice with water; 1% hydrochloric acid alcohol differentiated for 5 seconds and washed twice with water; eosin stained for 2 minutes and washed twice with water; neutral gum sealed and examined under a microscope. The results are as follows Figure 20As shown in Figures A and B, in C57BL / 6 and C57BL / 6 dirty cage mice, the results of histopathological examination showed that the engineered probiotic LC-pPG-CLA could significantly improve tissue damage caused by ETEC K88 infection, indicating that the engineered probiotic LC-pPG-CLA effectively alleviated the inflammatory damage caused by ETEC by inducing systemic and local mucosal immune responses.

[0092] 6. Effects of engineered probiotics on the intestinal flora of mice The feces of each group of C57BL / 6 mice after the last immunization were sequenced by 16S sequencing, and the data were analyzed on the Painuosen Gene Cloud Platform to detect the composition of the bacterial flora. Figure 19 As shown, in the intestinal microbial communities of C57BL / 6 mice, Bacteroidetes and Firmicutes accounted for a high proportion in all groups, representing the dominant bacterial groups in the mouse intestine. This suggests that mice immunized with engineered probiotics can modulate the relative abundance of Firmicutes and Bacteroidetes. Sequencing results from the dirty cage group demonstrate that probiotics can promote "immune training" through environmental changes.

[0093] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An anti-ETEC engineered probiotic, characterized in that: The anti-ETEC engineered probiotics are constructed by using lactic acid bacteria as a presentation vector, amplifying the CLA gene encoding linoleic acid isomerase from Lactobacillus rhamnosus, and connecting it with the lactobacillus expression vector pPG612 to construct the resulting engineered probiotic LC-pPG-CLA. The CLA nucleotide sequence is shown in SEQ ID NO.

3.

2. The method for constructing the engineered probiotic LC-pPG-CLA according to claim 1, characterized in that: The steps include: The pPG-CLA plasmid with the nucleotide sequence shown in SEQ ID NO.4 was electroporated into L. casei The engineered probiotic LC-pPG-CLA was obtained after culture in ATCC 393 competent state.

3. The method for constructing the engineered probiotic LC-pPG-CLA according to claim 2, characterized in that: The construction method of the pPG-CLA plasmid comprises the following steps: S1, amplification of CLA gene; The CLA gene encoding linoleic acid isomerase in Lactobacillus rhamnosus was amplified by PCR using the genome of Lactobacillus rhamnosus as a template, and the target gene was recovered. S2, construction of pEASY-CLA recombinant plasmid; The CLA target gene fragment recovered in the previous step was ligated with the cloning vector pEASY-Blunt-Zero; the ligation product was transformed into MC1061 competent cells and cultured to obtain the pEASY-CLA recombinant plasmid; S3, construction of the pPG-CLA recombinant plasmid, including the following steps: The Escherichia coli containing the pPG612 plasmid was activated and the plasmid was extracted; the recombinant plasmid pEASY-CLA and the expression vector pPG612 were double-digested respectively; after the digestion, agarose gel electrophoresis was performed to recover the target gene and vector fragment; the target gene and the expression vector pPG612 were ligated and transformed into MC1061 competent cells. After culture, the plasmid was extracted and double-digested and PCR identified. The suspected plasmid was sent for sequencing and named pPG-CLA after alignment.

4. The method for constructing the engineered probiotic LC-pPG-CLA according to claim 3, characterized in that: The enzymes for double digestion are restriction endonucleases SmaI and NcoI.

5. The method for constructing the engineered probiotic LC-pPG-CLA according to claim 3, characterized in that: The target gene and expression vector pPG612 were ligated using T4 DNA ligase.

6. Use of the engineered probiotic LC-pPG-CLA according to claim 1 in the preparation of anti-ETEC drugs.

7. Use of the engineered probiotic LC-pPG-CLA according to claim 6 in the preparation of anti-ETEC drugs, characterized in that: The drug has at least one of the following effects: (1) Improve the rapid weight loss and diarrhea symptoms caused by ETEC K88 infection; (2) Inhibit colon shortening symptoms caused by ETEC K88 infection; (3) Upregulate the expression levels of anti-inflammatory factor IL-10 and intestinal tight junction proteins Occludin-1, ZO-1, and p65 genes, maintaining intestinal epithelial cell permeability and intestinal barrier function; (4) Alleviate inflammatory damage caused by ETEC; (5) Down-regulate the gene expression levels of pro-inflammatory factors IL-1β and TNF-α; (6) Improve intestinal villus damage; (7) Increase the abundance of Firmicutes and Bacteroidetes, and reduce the relative abundance of harmful bacteria Proteobacteria.

8. Use of the engineered probiotic LC-pPG-CLA according to claim 1 in the preparation of anti-inflammatory bowel disease drugs and in the preparation of drugs for the prevention and / or treatment of enterotoxigenic Escherichia coli infections.

9. The use according to claim 8, characterized in that The medicine is an oral preparation, including tablets, capsules, oral liquid or granules.

10. The use according to claim 8, characterized in that The medicine is a vaccine.

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