Hsp60 receptor-targeted recombinant lactobacillus plantarum and construction method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and provides Hsp60 receptor targeted recombinant plant lactobacillus, a construction method and application. The plant lactobacillus is used as a host, pPG612 is used as an expression vector, and CsgB genes or CsgF genes are expressed. Two strains of engineering probiotics can express target proteins and can stably express on the surface of the bacterial body, effectively adhere to AB.9 intestinal epithelial cells, and can inhibit the adhesion and invasion of AB.9 cells. A. veronii Secondly, the engineering probiotics LP-pPG-CsgB and LP-pPG-CsgF can promote the proliferation of dendritic cells, T lymphocytes and B lymphocytes, and improve the secretion of intestinal mucus IgT. The engineering bacteria can also competitively bind to the Hsp60 receptor of intestinal epithelial cells, down-regulate the expression of p65 through the TLR4 / TLR5 dependent NF-κB signal pathway, inhibit the activation of the NF-κB signal pathway, regulate the redistribution of intestinal tight junction proteins, and down-regulate the expression of pro-inflammatory factors. Furthermore, the engineering bacteria can increase the abundance of intestinal beneficial flora of the snakehead, and the flora can mediate and activate the immune response through the feeding environment.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to Hsp60 receptor-targeting recombinant Lactobacillus plantarum, its construction method, and its application. Background Technology

[0002] Aeromonas vilvae ( Aeromonas veronii It can infect aquatic animals, including fish, as well as mammals, including humans, causing gastroenteritis, peritonitis, meningitis, sepsis, and wound infections in humans. This not only causes huge economic losses to the aquaculture industry but also seriously threatens human health.

[0003] The first line of defense against Aeromonas versicolor invading the body is the host's mucous membrane tissue. Studies have found that... A. veronii The adhesion protein (AP)-mediated pathway plays a crucial role in the invasion of the host epithelial barrier by pathogens. AP can specifically bind to the host cell surface receptor Hsp60 (human heat shock protein) to activate the NF-κB (nuclear factor κB) pathway. The aha and Inp genes can express adhesion proteins, thereby promoting the regulation of host immune function. The above research provides potential antigen gene selection for genetically engineered vaccines.

[0004] Two recombinant lactic acid bacteria strains, LP-pPG-CsgA and LP-pPG-CsgG, have been constructed, but novel Hsp60 receptor-targeting recombinant Lactobacillus plantarum strains still need to be developed to better reduce [the risk of infection]. A. veronii Its infectivity. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides an Hsp60 receptor-targeting recombinant *Lactobacillus plantarum*, its construction method, and its applications. This invention clones the CsgB and CsgF genes, which possess adhesion capabilities to *Aeromonas vesiculosus*, into a *Lactobacillus* expression vector, and then electroporates the recombinant plasmid into *Lactobacillus plantarum* (…). Lactobacillus plantarum Two engineered strains, LP-pPG-CsgB and LP-pPG-CsgF, were constructed using NC8, which effectively improved the ability to prevent and control Aeromonas verrucosa infections. In particular, the competitive invasion ability of the engineered strain LP-pPG-CsgF was significantly enhanced.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present application provides an Hsp60 receptor targeted recombinant Lactobacillus plantarum, which is Lactobacillus plantarum as a host, and expresses CsgB gene with nucleotide sequence as shown in SEQ ID NO. 3 or CsgF gene with nucleotide sequence as shown in SEQ ID NO. 4 by using pPG612 as an expression vector.

[0008] The Lactobacillus plantarum in the present application is not particularly limited in type and source, preferably, the Lactobacillus plantarum is Lactobacillus plantarum NC8. L. plantarum NC8 to obtain better antibacterial effect.

[0009] The second aspect of the present application provides a construction method of the Hsp60 receptor targeted recombinant Lactobacillus plantarum, comprising:

[0010] The CsgB gene with nucleotide sequence as shown in SEQ ID NO. 3 or the CsgF gene with nucleotide sequence as shown in SEQ ID NO. 4 is connected with the expression vector pPG612 to obtain the recombinant plasmid pPG-CsgB or pPG-CsgF.

[0011] The recombinant plasmid pPG-CsgB or pPG-CsgF is transformed into the Lactobacillus plantarum to obtain the strain LP-pPG-CsgB or LP-pPG-CsgF.

[0012] Further, the transformation method is electroporation, which realizes the introduction of the CsgB and CsgF genes with high efficiency and low toxicity.

[0013] The third aspect of the present application provides the application of the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum in the production of proteins.

[0014] Further, the protein is the protein expressed by the CsgB gene, and the CsgB gene is a gene encoding CsgB protein in Escherichia coli, which plays a key role in bacterial biofilm formation.

[0015] Further, the protein is the protein expressed by the CsgF gene, and the CsgF is an important chaperone, and the CsgF exposed on the outer membrane of the cell participates in the polymerization of CsgA regulated by CsgB.

[0016] Further, the method comprises inoculating the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum into MRS broth to ferment and produce the protein.

[0017] The fourth aspect of the present application provides the application of the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum in the preparation of an oral vaccine for preventing and treating Aeromonas veronii.

[0018] In a fifth aspect, the application provides use of the Hsp60 receptor-targeted recombinant Lactobacillus plantarum described above in the preparation of a medicament for treating inflammatory bowel disease.

[0019] Advantages of the application

[0020] (1) The application uses Lactobacillus plantarum (NC8) as a host to construct two types of engineering probiotics expressing pilus subunit CsgB and CsgF genes. Both of the two types of engineering probiotics can express target proteins and stably express on the bacterial surface, effectively adhere to AB.9 intestinal epithelial cells, and inhibit the adhesion and invasion of AB.9 cells. Lactobacillus plantarum Secondly, the engineering probiotics LP-pPG-CsgB and LP-pPG-CsgF can promote the proliferation of dendritic cells, T lymphocytes, and B lymphocytes, and increase the secretion of intestinal mucus IgT. The engineering bacteria can also competitively bind to the Hsp60 receptor of intestinal epithelial cells, down-regulate the expression of p65 through the TLR4 / TLR5-dependent NF-κB signaling pathway, inhibit the activation of the NF-κB signaling pathway, regulate the redistribution of intestinal tight junction proteins, down-regulate the expression of pro-inflammatory factors, and increase the abundance of beneficial bacteria in the intestine of the snakehead. The bacterial flora can mediate the activation of immune response through the feeding environment, resist infection, and provide an effective treatment approach for inflammatory bowel disease and other gastrointestinal diseases. A. veronii A. veronii

[0021] (2) Compared with the existing recombinant lactic acid bacteria LP-pPG-CsgA and LP-pPG-CsgG, the engineering probiotics LP-pPG-CsgB and LP-pPG-CsgF of the application have significantly enhanced ability to resist infection. A. veronii BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings, which form a part of this application, are included to provide a further understanding of the application and are incorporated in and constitute a part of this application. The embodiments of these drawings are set forth to explain the present application and do not constitute any limitation on the present application.

[0023] Figure 1 The figure is a model diagram of the engineering bacteria;

[0024] Figure 2 The figure is the growth curve detection result of the bacterial strain;

[0025] Figure 3 The figure is the detection result of the expressed proteins of the engineering bacteria LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB, and LP-pPG-CsgF;

[0026] Figure 4 The figure is the detection result of the growth performance of the engineering bacteria by plate counting method;

[0027] ​​​Figure 5 For A. veronii The results of the influence of AB.9 cell survival rate detection;

[0028] Figure 6 For the influence of the engineering bacteria on AB.9 cell survival rate detection results, (A) 2h, (B) 4h, (C) 6h, (D) 8h;

[0029] Figure 7 For the detection of the adhesion and invasion ability of the engineering bacteria to AB.9 cells, (A) adhesion ability detection, (B) competition adhesion ability of the engineering bacteria, (C) adhesion reduction rate of the engineering bacteria and pathogenic bacteria, (D) invasion ability detection, (E) competition invasion ability of the engineering bacteria, (F) invasion reduction rate of the engineering bacteria and pathogenic bacteria;

[0030] Figure 8 For the detection of the mRNA expression level of AB.9 cell related factors by the engineering bacteria, (A) AB.9 cell Hsp60 gene mRNA level detection results, (B) AB.9 cell IL-10 gene mRNA level detection results, (C) AB.9 cell IL-6 gene mRNA level detection results, (D) AB.9 cell IL-β gene mRNA level detection results, (E) AB.9 cell TNF-α gene mRNA level detection results;

[0031] Figure 9 For the detection of the mRNA level of (A) Occludin, (B) claudin-1, (C) ZO-1 gene in AB.9 cells by the engineering bacteria;

[0032] Figure 10 For the detection results of body weight changes of E. argus immunized by the engineering bacteria, the local enlarged view is the detection results of body weight changes at 120h;

[0033] Figure 11 For the detection results of the number of engineering bacteria in the intestine;

[0034] Figure 12 For the detection results of (A) CD80 + CD11C + DCs cell proportion, (B) CD86 + CD11C + DCs number in intestinal tissue;

[0035] Figure 13 For the detection results of B220 + IgT + B cell number in intestinal tissue and spleen, (A) intestinal tissue; (B) spleen;

[0036] Figure 14 For the detection results of CD4 + CD8+ T cell number detection results, (A) intestinal tissue; (B) spleen;

[0037] Figure 15 For intestinal tissue and spleen CD3 + CD4 + T cell number detection results, (A) intestinal tissue; (B) spleen;

[0038] Figure 16 For the detection results of the mRNA transcription level of IL-10 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0039] Figure 17 For the detection results of the mRNA transcription level of IL-6 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0040] Figure 18 For the detection results of the mRNA transcription level of IL-1β gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0041] Figure 19 For the detection results of the mRNA transcription level of TNF-α gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0042] Figure 20 For the detection results of the mRNA transcription level of TLR4 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0043] Figure 21 For the detection results of the mRNA transcription level of TLR5 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0044] Figure 22 For the detection results of the mRNA transcription level of MyD88 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0045] Figure 23 For the detection results of the mRNA transcription level of Hsp60 gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0046] Figure 24 For the detection results of the mRNA transcription level of IκB-α gene in the intestine of Channa argus, (A) detection results after the first immunization; (B) detection results after the booster immunization;

[0047] Figure 25The mRNA transcription level detection results of p53 gene in the intestine of snakehead are (A) detection results after first immunization; (B) detection results after booster immunization;

[0048] Figure 26 The influence of the engineering bacteria on the expression of (A) Hsp60, (B) p53, (C) P-p53, (D) p65 and (E) P-p65 proteins in the intestine of snakehead and (F) expression detection results;

[0049] Figure 27 The influence of the engineering bacteria on the expression of (A) Occludin, (B) Claudin1, (C) ZO-1 and (D) E-cadherin proteins in the intestine of snakehead and (E) expression detection results;

[0050] Figure 28 The classification results of the intestinal flora species composition of snakehead;

[0051] Figure 29 The histopathological detection results of the intestine of snakehead after challenge;

[0052] In the above figures, ns represents no significant difference, * represents P value < 0.05: ** represents P value < 0.01, *‌** represents P value < 0.001, and *‌*** represents P value < 0.0001;

[0053] Control is a blank control group, A. veronii For A. veronii TH0426, LP wt For L. plantarum NC8, LP-pPG-aha is the engineering bacteria LP-pPG-aha, LP-pPG-Inp is the engineering bacteria LP-pPG-Inp, LP-pPG-CsgB is the engineering bacteria LP-pPG-CsgB, and LP-pPG-CsgF is the engineering bacteria LP-pPG-CsgF. DETAILED DESCRIPTION

[0054] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0055] The application will be described in further detail below with specific embodiments. It should be noted that the specific embodiments are illustrative of the application and are not limiting.

[0056] The source of all biological materials (genes, vectors, microorganisms, etc.):

[0057] L. plantarumNCBI Reference Sequence of NC8: NZ_AGRI01000002.1, A. veronii NCBI database information of TH0426: BioProject: PRJNA293940, BioSample: SAMN04012505, Lactobacillus casei anchoring expression vector pPG612 was purchased from BNCC company; pEASYBlunt cloning vector was purchased from Beijing Zison Gold Biotechnology Co., Ltd.

[0058] Example 1: amplification of aha, Inp, CsgB, CsgF genes and construction of recombinant plasmid

[0059] The construction method is shown in Figure 1 , and specifically includes:

[0060] 1. Acquisition and amplification of target genes:

[0061] The strain A. veronii TH0426 was activated in LB broth at 37°C for 8h, and after three generations, it was streaked on LB agar plates and cultured at 37°C for 12h. Single colonies were picked and purified. The purified bacterial solution was used to extract the genome.

[0062] According to the A. veronii TH0426 whole genome data (CP012504.1) registered in GenBank, the gene sequences of aha, Inp, CsgB and CsgF of the strain (SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4) were obtained. Four pairs of specific primers for connecting with the Lactic acid bacteria expression vector pPG612 were designed, and His tag sequence was added. They are: aha gene fragment (F1, R1) containing Sma I, EcoR V restriction site at both ends, Inp gene fragment (F2, R2) containing Nco I, Sma I restriction site at both ends, CsgB gene fragment (F3, R3) containing Nco I, Not I restriction site at both ends, and CsgF gene fragment (F4, R4) containing Nco I, Sac II restriction site at both ends. The primers were synthesized by ChunChun Kumei Biological Company.

[0063] The expected fragment sizes of the primers are aha gene 1038 bp, Inp gene 381 bp, CsgB gene 1590 bp, and CsgF gene 840 bp. The extracted A. veroniiThe four pairs of primers were amplified respectively with TH0426 genome as template. The amplification conditions were as follows: pre-denaturation (95°C, 5 min); denaturation (94°C, 1 min), annealing (aha: 53°C, Inp: 68°C, CsgB: 68°C, CsgF: 68°C, 1 min), extension (72°C, 1 min) for 30 cycles; and re-extension (72°C, 10 min). After the PCR amplification, 5 μL of the product was taken for electrophoresis detection, and the results were observed by a gel imaging instrument. The target genes were recovered by an OMEGA gel recovery kit, and were stored at -20°C.

[0064] The PCR reaction system was as follows: PrimeSTAR Max 10 μL, upstream primer 1 μL, downstream primer 1 μL, template 1 μL, ddH2O (double distilled water) 7 μL, and the total system was 20 μL.

[0065] 2. The frozen strain was used E. coli The MC1061 was used to prepare competent cells.

[0066] 3. Construction and identification of the cloning vector:

[0067] The aha, Inp, CsgB and CsgF gene fragments recovered by the gel were connected to the pEASY-Blunt cloning vector respectively, and the reaction was performed at 25°C for 4 h. The connection system was as follows: pEASYBlunt Vector 1 μL, target gene 4 μL, and the reaction system was 5 μL.

[0068] The connection product was transformed into the MC1061 competent cells by the ordinary transformation method, which included the following steps:

[0069] (1) The competent cells E. coli The MC1061 was placed in an ice bath for 15 min, and after recovery, 50 μL was mixed with 5 μL of the connection product, and was placed in an ice bath for 30 min;

[0070] (2) 42°C heat shock for 90 s, and ice bath for 3 min 40 s;

[0071] (3) 800 μL of LB broth was added, and was cultured at 37°C on a shaker for 2.5 h;

[0072] (4) After the bacterial solution was turbid, it was centrifuged at 5000 rpm for 8 min, and the supernatant was discarded. 100 μL of the resuspended bacterial body was coated on an LB agar plate (50 μg / mL of ampicillin solution), and was cultured at 37°C for 16 h to appear colonies. A single colony was picked for PCR identification, and the suspected plasmid was sent for sequence detection. After the identification was correct, they were named as pEASYBlunt-aha, pEASYBlunt-Inp, pEASYBlunt-CsgB and pEASYBlunt-CsgF respectively.

[0073] 4. Expression vector and target gene cloning:

[0074] The pPG612, pEASYBlunt-aha, pEASYBlunt-Inp, pEASYBlunt-CsgB, and pEASYBlunt-CsgF plasmids were respectively double-digested, and the enzyme reaction system was as follows: template (plasmid) 25 μL, buffer (10x) 5 μL, restriction enzyme (EcoRI) 1.5 μL, restriction enzyme (HindIII) 1.5 μL, and ddH2O 17 μL. EcoR V / Nco I / Sac II) 1.5 μL, restriction enzyme (EcoRI) Sma I / Not I) 1.5 μL, ddH2O 17 μL.

[0075] 5. Connection of expression vector and target gene sequence:

[0076] The pPG612 and target gene were connected by T4 DNA ligase, and the connection product was transferred into E. coli MC1061 competence. The reaction system was incubated at 16°C overnight, and the bacterial solution cultured for 2.5 h was coated on an LB agar plate (chloramphenicol concentration Cm: 10 μg / mL). After single colonies were picked and preliminarily identified by PCR and enzyme digestion, the suspected strains were sent for sequence detection. The strain with correct sequence was named as pPG-aha, pPG-Inp, pPG-CsgB, and pPG-CsgF.

[0077] The T4 connection reaction system was T4 DNA ligase (ligase) 2 μL, T4 DNA ligase buffer (10x) 1 μL, target gene fragment 5.5 μL, and vector fragment 1.5 μL, and the reaction system was 10 μL.

[0078] Example 2: Construction and verification of recombinant lactic acid bacteria

[0079] 1. Preparation L. plantarum NC8 competence:

[0080] (1) The constructed strain was inoculated in liquid MRS broth, and after three generations, it was streaked on an MRS agar plate, and single colonies were picked and purified;

[0081] (2) The purified bacterial solution was inoculated in 200 mL MRS broth at a proportion of 2wt%, and was anaerobically cultured at 37°C to OD 600 nm=0.6 (6 h);

[0082] (3) Transfer the cultured bacterial solution to a sterile 50mL centrifuge tube, incubate on ice for 30min, then centrifuge at 4℃ and 4000rpm for 15min and discard the supernatant;

[0083] (4) Resuspend the bacterial precipitate twice with 20 mL of pre-cooled EPWB (0.0936 g NaH2PO4·2H2O and 0.0213 g MgCl2·6H2O, ddH2O to a final volume of 1 L), centrifuge at 4℃ and 4000 rpm for 15 min, and discard the supernatant;

[0084] (5) After collecting the bacterial cells, gently suspend the precipitate with 20 mL of pre-cooled EPB (200 mL EPWB and 20.53782 g sucrose), centrifuge at 4℃ and 4000 rpm for 15 min, and discard the supernatant;

[0085] (6) Resuspend the bacterial pellet in 1 mL of pre-cooled EPB, incubate on ice for 20 min, and dispense 100 μL / tube into pre-cooled 1.5 mL centrifuge tubes and store at -80 °C.

[0086] 2. Electroconversion:

[0087] The correctly sequenced recombinant plasmid was transformed into [a specific material] via electroporation. L. plantarum NC8 competent cells include:

[0088] (1) L. plantarum NC8 competent cells were revived by incubating on ice for 15 min, and then 200 μL of each cell was mixed with 10 μL of the constructed recombinant plasmid and incubated on ice for 10 min.

[0089] (2) Pre-cool the electrocup to 4°C, transfer the mixture to the electrocup (2mm), and place in an ice bath for 10 minutes;

[0090] (3) Electric shock conditions: 2.52kV, 5ms;

[0091] (4) After the electric shock is completed, add 900 μL of preheated MRS broth (containing 15 wt% sucrose), stabilize in an ice bath for 20 min, and then place in an anaerobic incubator at 37 ℃ for 3 h.

[0092] (5) Take 100 μL of turbid bacterial solution and spread it on an MRS agar plate (Cm: 10 μg / mL) and anaerobic at 37℃ for 36 h;

[0093] 3. Validation of recombinant lactic acid bacteria gene levels

[0094] The next day, single colonies were picked into 10 mL MRS broth (Cm: 5 pg / mL) and incubated anaerobically at 37°C overnight; 4 mL of bacterial solution was used to extract plasmids. The small plasmid extraction kit was used to resuspend 250 pL P1 solution, then 250 pL lysozyme (100 mg / mL) was added, and the solution was treated at 37°C for 1 h. According to the instructions, PCR and double enzyme digestion were performed on the recombinant plasmids, and the suspected plasmids were sent for detection. The correct strains were named LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB, and LP-pPG-CsgF, and were stored at -40°C.

[0095] Example 3: Verification of recombinant lactic acid bacterial proteins

[0096] 1. Determination of the growth curve of the engineered bacteria;

[0097] 2. Preparation of recombinant bacterial proteins:

[0098] The recombinant bacteria were inoculated in MRS liquid medium (Cm: 5 pg / mL), and after 2 generations, they were inoculated in MRS broth (Cm: 5 pg / mL and 2 wt% lactose) at a ratio of 3% and induced at 37°C anaerobically for 20 h or more. The induced bacterial solution was washed with 2 mL of sterile PBS (phosphate buffered solution) three times, and each time it was centrifuged (4°C, 8000 rpm, 12 min). After resuspension in 1 mL of PBS, 500 pL of lysozyme was added for wall breaking (37°C, 1 h), and after wall breaking, the solution was centrifuged (4°C, 8000 rpm, 12 min) to collect the precipitate. Protein quantification was performed using a BCA kit, and the solution was diluted with sterile PBS to 10 pg / pL. 400 pL of the resulting protein was mixed with 100 pL of SDS-PAGE buffer (5x), boiled at 100°C for 8 min, and then stored at -80°C after denaturation.

[0099] 3. SDS-PAGE:

[0100] The separation gel and the concentrated gel were prepared according to the preparation box of Beijing Sunshine Gel (gel spacing 1 mm).

[0101] 4. Western blot and indirect immunofluorescence.

[0102] Example 4: Analysis of the biological characteristics of Hsp60 receptor-targeted engineered bacteria

[0103] 1. Resuscitation, medium change, subculture, and cryopreservation of AB.9 cells;

[0104] 2. Culture of bacteria:

[0105] (1) A. veronii Preparation of TH0426 bacterial solution

[0106] a. Selection A. veroniiThe TH0426 plateau phase bacterial culture was washed three times with pre-cooled PBS, pre-cooled at 4°C, centrifuged at 5000 rpm for 10 min, and the bacterial cells were collected.

[0107] b. Adjust the concentration to 1.5 × 10⁻⁶ using complete culture medium. 7 CFU / mL, to be used during cell incubation.

[0108] (2) Preparation of engineered bacteria culture

[0109] a. Based on the growth curve of the engineered bacteria obtained in Example 3, select the plateau phase bacterial solution, wash it three times with pre-cooled PBS, pre-cool it at 4°C in a centrifuge, centrifuge it at 5000 rpm for 10 min, and collect the bacterial cells;

[0110] b. Adjust the concentration to 1.5 × 10⁻⁶ using complete culture medium. 7 CFU / mL, to be used after incubation with cells.

[0111] 3. Detection of the optimal interaction time between bacteria and AB.9 cells:

[0112] AB.9 cells were divided into 1×10 5 Inoculate cells at a concentration of [number] cells / mL into 24-well plates, adding 1 mL of complete culture medium to each well for resuscitation. Incubate at 25°C in a 5% CO2 incubator. Change the medium after 24 hours until the cells have confluently colonized. Once confluent, discard the complete culture medium and wash three times with pre-warmed PBS to remove any floating dead cells. Then, inoculate the cells at a fold increase in infection (MOI) of 100:1. A. veronii TH0426 and AB.9 cells were co-cultured at 25°C in a 5% CO2 incubator for 2 h, 4 h, 6 h, and 8 h. After co-culture, the culture medium was discarded, and the cells were washed with pre-warmed PBS. Cells were then digested with trypsin containing EDTA, and the single-cell suspension was appropriately diluted and mixed with trypan blue solution at a 9:1 (volume ratio). The mixture was added to a red blood cell counting chamber, and cell viability was immediately assessed under an inverted microscope. Cells not co-cultured with bacteria served as controls, with three replicates per group. Live cells were transparent and colorless, while dead cells appeared distinctly blue.

[0113] 4. Recombinant lactic acid bacteria and A. veronii Assay of TH0426's ability to adhere to AB.9 cells:

[0114] (1) The four engineered strains and the wild strain were constructed. L. plantarum NC8 and A. veronii TH0426 activation was specifically performed as follows: the engineered bacteria were anaerobically cultured in MRS liquid (Cm: 5 μg / mL), and the wild-type LP strain was anaerobically cultured in ordinary MRS liquid medium. A. veronii TH0426 was cultured in standard LB liquid medium;

[0115] (2) Take the platform bacteria liquid, wash three times with pre-cooled PBS, centrifuge pre-cooled at 4℃, 5000 rpm for 10 min, collect the bacteria, and resuspend with MEM incomplete medium. According to the infection multiple (MOI) 100:1, inoculate the bacteria liquid into the single layer of cells in the 6-hole plate (1.5×10 7 bacteria and 1.5×10 5 cells);

[0116] (3) After 6h of culture at 25℃ in a 5% CO2 incubator (4h of culture), discard the complete culture medium, and wash the cells three times with pre-warmed PBS to wash away the unadherent bacteria. Be gentle when washing to avoid cell shedding; A. veronii

[0117] (4) After washing, add 200μL of 1% Triton X-100 to each hole at 25℃ for 30 min, dilute the lysis product with PBS at a ratio of 1:10, and spread it on MRS solid medium (recombinant bacteria MRS medium containing Cm: 10μg / mL) to count the number of adherent bacteria. Incubate at 37℃ in an anaerobic incubator overnight. Each group has three repeats to reduce errors. Adhesion rate = adherent bacteria number / total bacteria number×100%.

[0118] 5. Detection of competition adhesion ability of the engineered bacteria A. veronii

[0119] (1) Adjust the four kinds of recombinant lactic acid bacteria and A. veronii TH0426 to 1.5×10 7 CFU / mL with incomplete medium;

[0120] (2) Co-infect the single layer of AB.9 cells with the four kinds of recombinant lactic acid bacteria and A. veronii TH0426 for 4h (25℃, 5% CO2 culture), and the Control is the blank control group, with PBS buffer added as the reagent;

[0121] (3) Wash the cells three times with pre-warmed PBS to wash away the unadherent bacteria. After washing, add 200μL of 1% Triton X-100 to each hole at 25℃ for 30 min, and dilute the four kinds of recombinant lactic acid bacteria and A. veronii TH0426 with PBS and count them with a plate. Calculate the adhesion rate and the adhesion reduction rate respectively. Competition adhesion rate = competition adherent bacteria number / total bacteria number×100%, and adhesion reduction rate = adhesion rate-competition adhesion rate.

[0122] 6. Detection of AB.9 cell invasion ability of the recombinant lactic acid bacteria and A. veronii TH0426

[0123] (1) Prepare​​A. veronii TH0426 bacteria solution and four kinds of recombinant lactic acid bacteria suspensions;

[0124] (2) After washing, 200 μL of incomplete medium (containing gentamicin: 200 μg / mL) was added for 2 h of continuous culture to completely kill the extracellular bacteria;

[0125] (3) At the end of the culture, the cells were washed three times with pre-warmed PBS, and 200 μL of 1% Triton X-100 was added for 30 min of lysis;

[0126] (4) After dilution with PBS, the cells were plated on plates (recombinant bacteria MRS agar containing Cm: 10 μg / mL, A. veronii TH0426 was a general LB agar plate), and the number of invasive bacteria was counted. The invasion rate = the number of invasive bacteria / the total number of bacteria x 100%.

[0127] 7. Competition invasion ability detection of the engineered bacteria A. veronii

[0128] The monolayer cells were co-infected with four kinds of recombinant lactic acid bacteria and A. veronii TH0426 for 4 h, and after the cells were washed three times with pre-warmed PBS, 200 μL of incomplete medium containing gentamicin was added for 2 h of culture. The competition invasion rate and the invasion reduction rate were calculated. The competition invasion rate = the number of competition invasive bacteria / the total number of bacteria x 100%, and the invasion reduction rate = the invasion rate - the competition invasion rate.

[0129] 8. Detection of the mRNA expression level of AB.9 cell-related factors by the engineered bacteria

[0130] Example 5: Evaluation of the immune effect of the Hsp60 receptor-targeted engineered bacteria

[0131] 1. Immunization scheme of the test animals:

[0132] (1) The snakeheads were randomly divided into a Control group, an LP wt group, an LP-pPG-aha group, an LP-pPG-Inp group, an LP-pPG-CsgB group, and an LP-pPG-CsgF group, with 50 fish in each group. The Control group and the LP wt group were used as control groups, and after two weeks of pre-feeding, the fish were orally immunized with probiotics according to the scheme shown in Table 1. The fish were fasted for 2-4 h before immunization and were normally fed 1-2 h after immunization. The fish were immunized once every week, and each time of immunization lasted for 5 days, and the fish were immunized twice in total. That is, the fish were immunized for the first time on day 1-5 of the formal test, and were immunized for the second time on day 13-17 after one week of interval. After each immunization, flow cytometry detection was performed. After the second immunization, 3 fish were randomly selected from each group to collect feces and intestinal contents for 16S rRNA bacterial flora diversity detection. ​

[0133] Table 1 Grouping of test animals

[0134]

[0135] (2) Check and collect feces and intestinal contents, and use MS-222 reagent (250 mg / L, Sigma, USA) to anesthetize and kill each group of snakeheads, disinfect the fish body surface with 75% alcohol by volume to remove bacteria, collect the intestinal tissue of the snakehead after dissection, and wash the attached blood with sterile PBS, freeze in liquid nitrogen, and store at -80°C.

[0136] (3) Detect the engineering bacteria by flow cytometry.

[0137] 2. Preparation of cell suspension;

[0138] 3. DC detection;

[0139] 4. B cell detection;

[0140] 5. T cell detection;

[0141] 6. Detection of intestinal colonization of recombinant lactic acid bacteria:

[0142] After the first oral immunization, on the 6th day, the intestinal colonization of the bacterial flora was detected, starting from the 24th hour after fasting to the 72nd hour, every 12 hours, a total of 5 times, 3 tails each time. Intestinal tissue was taken by dissection, frozen in liquid nitrogen, and then ground into powder. The ground tissue was then transferred to a sterile centrifuge tube containing 1 mL of sterile PBS buffer. After dilution, the plate count was performed, and 3 replicates were set, and random colonies were selected for PCR verification.

[0143] 7. Detection of cytokine mRNA expression level;

[0144] 8. Detection of the effect of engineering bacteria on the expression of intestinal related proteins of snakeheads;

[0145] 9. Take the intestinal and spleen tissues of each group of snakeheads after challenge, and perform histopathological detection;

[0146] 10. 16S microbial diversity detection:

[0147] Fresh feces and intestinal contents of each group of snakeheads after 18 days of immunization were detected for microbial diversity. The sequence was detected for species composition, and the intestinal microbial diversity was analyzed by Alpha and Beta diversity analysis on the Psonnelo gene cloud platform.

[0148] The experimental results show that:

[0149] For example Figure 2As shown, the growth rate of the engineering bacteria was basically consistent with that of the wild strain, and there was no obvious difference, which indicated that the expression of the target protein had no obvious influence on the growth of the host strain. As shown in Figure 3 As shown, it was proved that the protein could be stably expressed in NC8; as shown in L. plantarum As shown, compared with NC8, the growth performance of the engineering bacteria was better, as shown in Figure 4 As shown, compared with the control group, L. plantarum The treatment time was 5h, and the cell survival rate was significantly lower than that of the control group (P<0.01). Figure 5 The treatment time was 5h, and the cell survival rate was significantly lower than that of the control group (P<0.01). A. veronii Figure 6 The treatment time was 8h, and the cell survival rate was significantly lower than that of the control group. L. plantarum As shown in (A, B, C) in the middle, compared with other strains, the adhesion and competitive adhesion ability of the engineering bacteria LP-pPG-CsgB and the engineering bacteria LP-pPG-CsgF were better,

[0150] As shown in (D, E, F) in the middle, the invasiveness and competitive invasiveness of the engineering bacteria LP-pPG-CsgF were better than Figure 7 A. veronii

[0151] As shown in (D, E, F) in the middle, the invasiveness and competitive invasiveness of the engineering bacteria LP-pPG-CsgF were better than Figure 7 A. veronii A. veronii

[0152] Figure 8 The mRNA expression level of AB.9 cell related factors detected by engineering bacteria, (A) AB.9 cell Hsp60 gene mRNA level detection result, (B) AB.9 cell IL-10 gene mRNA level detection result, (C) AB.9 cell IL-6 gene mRNA level detection result, (D) AB.9 cell IL-1β gene mRNA level detection result, (E) AB.9 cell TNF-α gene mRNA level detection result; The expression amount of Hsp60 gene, IL-10 gene of the engineering bacteria LP-pPG-CsgB and LP-pPG-CsgF group was significantly higher than that of the blank control group, and the IL-1β gene, IL-6 gene and TNF-α gene of the engineering bacteria LP-pPG-CsgB and LP-pPG-CsgF group were lower than those of the blank control group.

[0153] Figure 9 ​​​​​​The results show the mRNA levels of (A) Occludin, (B) claudin-1, and (C) ZO-1 genes in AB.9 cells by engineered bacteria; the expression levels of Occludin, claudin-1, and ZO-1 genes were upregulated in the recombinant lactic acid bacteria group.

[0154] Figure 10 The image shows the weight change of snakehead fish after immunization with engineered bacteria. A magnified view shows the weight change at 120 hours. The weight of the LP-pPG-CsgB and LP-pPG-CsgF groups was better than that of the LP group. wt Group;

[0155] Figure 11 Results of the detection of the number of engineered bacteria colonizing the intestines; Figure 12 (A)CD80 in intestinal tissue + CD11C + DCs cell ratio, (B)CD86 + CD11C + DCs count results; the engineered probiotics LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF constructed in this invention can increase CD80 after immunizing snakehead fish. + Cells and CD86 + The cell count indicates that the constructed targeted engineered bacteria can promote the differentiation and maturation of dendritic cells (DCs), thereby promoting the immune system's clearance of intestinal pathogens. Figure 13 It is B220 in intestinal tissue and spleen + IgT + B cell count results, (A) intestinal tissue; (B) spleen; Figure 14 CD4 is found in intestinal tissue and spleen. + CD8 + T cell count results, (A) intestinal tissue; (B) spleen; Figure 15 CD3 is found in intestinal tissue and spleen. + CD4 + The results of T cell count detection (A) intestinal tissue; (B) spleen; LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB, and LP-pPG-CsgF can all stimulate the proliferation of immune-related cells in the intestine and spleen. This indicates that orally administered engineered bacteria can be taken up and processed by dendritic cells (DCs), stimulating T cells and B cells to exert cellular and humoral immunity, laying the foundation for the development of oral probiotic vaccines.

[0156] Figure 16 The results are the mRNA transcription levels of the IL-10 gene in the intestine of snakehead fish, (A) results after the first immunization; (B) results after the booster immunization; Figure 17are the detection results of mRNA transcription level of IL-6 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 18 are the detection results of mRNA transcription level of IL-1β gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 19 are the detection results of mRNA transcription level of TNF-α gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 20 are the detection results of mRNA transcription level of TLR4 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 21 are the detection results of mRNA transcription level of TLR5 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 22 are the detection results of mRNA transcription level of MyD88 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 23 are the detection results of mRNA transcription level of Hsp60 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 24 are the detection results of mRNA transcription level of IκB-α gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 25 are the detection results of mRNA transcription level of p53 gene in the intestine of Ophicephalus argus, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 26 are the influence of the engineering bacteria on the protein expression of (A) Hsp60, (B) p53, (C) P-p53, (D) p65 and (E) P-p65 in the intestine of Ophicephalus argus and (F) expression detection results; Figure 27are the effects of the engineering bacteria on the expression of (A) Occludin, (B) Claudin1, (C) ZO-1 and (D) E-cadherin proteins in the intestine of snakehead and (E) expression detection results; in the real-time fluorescence quantitative PCR detection, it is found that LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF can promote the expression of Hsp60 gene of snakehead, indicating that the adhesion proteins expressed by the engineering bacteria can specifically bind to the Hsp60 receptor of snakehead intestinal epithelial cells. IκB-α plays a key inhibitory role in the NF-κB signal transduction pathway, and p53 can also have an inhibitory effect, thereby promoting cell apoptosis and inhibiting inflammation. Compared with the control group, the LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF groups can promote the expression of IκB-α gene, thereby promoting the induction and secretion of various anti-inflammatory cytokines. Immune cells and tissue epithelial cells can secrete IL-10 anti-inflammatory factors, which can reduce inflammatory damage by inhibiting the excessive production of inflammatory cytokines. The present application finds that after oral immunization of engineering bacteria, the anti-inflammatory factors of each group of snakehead are continuously increased, indicating that the constructed LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF can inhibit tissue inflammatory damage through the secretion of anti-inflammatory cytokines. At the same time, as shown in Figure 28 、 Figure 29 , the engineering bacteria can increase the abundance of beneficial bacteria in the intestine of snakehead, and the bacterial flora can mediate and activate the immune response through the transmission of the feeding environment, resist A. veronii infection, and provide an effective treatment for inflammatory bowel disease and other gastrointestinal diseases.

[0157] The above only describes the preferred embodiments of the present application and is not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A Hsp60 receptor-targeted recombinant Lactobacillus plantarum, characterized in that, Lactobacillus plantarum is used as a host and pPG612 is used as an expression vector to express CsgB gene with nucleotide sequence as shown in SEQ ID NO. 3 or CsgF gene with nucleotide sequence as shown in SEQ ID NO. 4; The Lactobacillus plantarum is Lactobacillus plantarum L. plantarum NC8.

2. A method of constructing a Hsp60 receptor-targeted recombinant plant lactobacillus, characterized by, comprising: CsgB gene with nucleotide sequence as shown in SEQ ID NO. 3 or CsgF gene with nucleotide sequence as shown in SEQ ID NO. 4 is connected with expression vector pPG612 to obtain recombinant plasmid pPG-CsgB or pPG-CsgF; The recombinant plasmid pPG-CsgB or pPG-CsgF is transformed into Lactobacillus plantarum to obtain strain LP-pPG-CsgB or LP-pPG-CsgF.

3. The method for constructing Hsp60 receptor-targeted recombinant plant lactobacillus according to claim 2, wherein, The transformation method is electroporation.

4. The application of Hsp60 receptor-targeting recombinant Lactobacillus plantarum in the production of proteins according to claim 1.

5. Use of the Hsp60 receptor-targeted recombinant Lactobacillus plantarum of claim 4 for the production of proteins, characterized by, The protein is the protein expressed by CsgB gene.

6. Use of the Hsp60 receptor-targeted recombinant Lactobacillus plantarum of claim 4 for the production of proteins, characterized by, The protein is the protein expressed by CsgF gene.

7. Use of the Hsp60 receptor-targeted recombinant plantarum lactis bacterium according to claim 4 for the production of proteins, characterized in that, comprising: The Hsp60 receptor-targeting recombinant Lactobacillus plantarum according to claim 1 is inoculated into MRS broth medium to ferment and produce proteins.

8. The application of Hsp60 receptor-targeting recombinant Lactobacillus plantarum in the preparation of oral vaccine for preventing and treating Veillonella wautersi according to claim 1.

9. The application of Hsp60 receptor-targeting recombinant Lactobacillus plantarum in the preparation of drugs for treating inflammatory bowel disease according to claim 1.

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