Hsp60 receptor targeted recombinant lactobacillus plantarum as well as construction method and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and provides Hsp60 receptor targeted recombinant lactobacillus plantarum as well as a construction method and application thereof, lactobacillus plantarum is taken as a host, pPG612 is taken as an expression vector, and CsgB gene or CsgF gene is expressed. The two engineering probiotics can express target protein and can be stably expressed on the surfaces of thalli, the AB.9 intestinal epithelial cells can be effectively adhered, and the adhesion and invasion of the A.veronii to the AB.9 cells can be inhibited. And 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 IgT secretion of intestinal mucus. The engineering bacteria can be competitively combined with an intestinal epithelial cell Hsp60 receptor, down-regulate p65 expression, inhibit NF-kappa B signal channel activation, regulate intestinal tight junction protein redistribution and down-regulate proinflammatory factor expression through a TLR4 / TLR5 dependent NF-kappa B signal channel, and the engineering bacteria can increase the abundance of effective microbial communities in the intestinal tract of the snakeheaded fish, so that the growth of the snakeheaded fish is promoted, and the growth of the snakeheaded fish is promoted. And the flora can transfer, mediate and activate immune response through a 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: In a first aspect, the present invention provides an Hsp60 receptor-targeting recombinant Lactobacillus plantarum, using Lactobacillus plantarum as the host and pPG612 as the expression vector, to express the CsgB gene with the nucleotide sequence shown in SEQ ID NO.3 or the CsgF gene with the nucleotide sequence shown in SEQ ID NO.4.

[0007] The present application does not make special limitation to the type and source of Lactobacillus plantarum, preferably, the Lactobacillus plantarum is Lactobacillus plantarum L. plantarum NC8, so as to obtain better antibacterial effect.

[0008] In the second aspect of the present application, a construction method of Hsp60 receptor targeted recombinant Lactobacillus plantarum is provided, comprising: The CsgB gene with the nucleotide sequence as shown in SEQ ID NO. 3 or the CsgF gene with the 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. The recombinant plasmid pPG-CsgB or pPG-CsgF is transformed into Lactobacillus plantarum to obtain the strain LP-pPG-CsgB or LP-pPG-CsgF.

[0009] Further, the transformation method is electrotransformation, which realizes the introduction of CsgB and CsgF genes with high efficiency and low toxicity.

[0010] In the third aspect of the present application, the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum is applied in the production of proteins.

[0011] Further, the protein is the protein expressed by the CsgB gene, and the CsgB gene is a gene encoding CsgB protein in Escherichia coli and plays a key role in bacterial biofilm formation.

[0012] 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 cells participates in the polymerization of CsgA regulated by CsgB.

[0013] Further, the method comprises inoculating the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum into MRS broth to ferment and produce proteins.

[0014] In the fourth aspect of the present application, the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum is applied in the preparation of an oral vaccine for preventing and treating Aeromonas veronii.

[0015] In the fifth aspect of the present application, the above-mentioned Hsp60 receptor targeted recombinant Lactobacillus plantarum is applied in the preparation of a drug for treating inflammatory bowel disease.

[0016] Advantages of the present application (1) The present application uses Lactobacillus plantarum (Lactobacillus plantarum) as a carrier to target the Hsp60 receptor of the host, so as to realize the targeted delivery of the CsgB gene and the CsgF gene to the host. Lactobacillus plantarumNC8 as the host, two kinds of engineering probiotics expressing pilus subunit CsgB and CsgF genes are constructed, both of the two kinds of engineering probiotics can express target proteins and can be stably expressed on the surface of bacterial bodies, can effectively adhere to AB.9 intestinal epithelial cells, and can inhibit A. veronii adhesion and invasion of AB.9 cells. Secondly, the engineering probiotics LP-pPG-CsgB and LP-pPG-CsgF can promote proliferation of dendritic cells, T lymphocytes and B lymphocytes, and increase intestinal mucus IgT secretion. The engineering bacteria can also competitively bind to Hsp60 receptors of intestinal epithelial cells, down-regulate p65 expression through a TLR4 / TLR5-dependent NF-κB signaling pathway, inhibit activation of the NF-κB signaling pathway, regulate redistribution of intestinal tight junction proteins, down-regulate expression of proinflammatory factors, and make the abundance of intestinal beneficial bacteria of Channa argus increase, and the bacterial flora can mediate activation of immune response through feeding environment, and resist A. veronii infection, thereby providing an effective treatment approach for inflammatory bowel disease and other gastrointestinal diseases.

[0017] (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 present application have significantly enhanced ability to resist A. veronii infection. BRIEF DESCRIPTION OF DRAWINGS

[0018] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the exemplary embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0019] Figure 1 It is a mode diagram for the engineering bacteria; Figure 2 It is a growth curve detection result of the bacterial strain; Figure 3 It is a detection result of the expressed proteins of the engineering bacteria LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF; Figure 4 It is a detection result of the growth performance of the engineering bacteria by plate counting method; Figure 5 It is A. veronii a detection result of the influence on the survival rate of AB.9 cells; Figure 6 It is a detection result of the influence of the engineering bacteria on the survival rate of AB.9 cells; (A) 2h, (B) 4h, (C) 6h, (D) 8h; Figure 7For the detection of the adhesion and invasion abilities 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, and (F) invasion reduction rate of the engineering bacteria and pathogenic bacteria; Figure 8 For the detection of the mRNA expression levels of AB.9 cell-related factors by the engineering bacteria, (A) mRNA level detection result of the AB.9 cell Hsp60 gene, (B) mRNA level detection result of the AB.9 cell IL-10 gene, (C) mRNA level detection result of the AB.9 cell IL-6 gene, (D) mRNA level detection result of the AB.9 cell IL-β gene, and (E) mRNA level detection result of the AB.9 cell TNF-α gene; Figure 9 For the mRNA level detection results of (A) Occludin, (B) claudin-1, and (C) ZO-1 genes in AB.9 cells by the engineering bacteria; Figure 10 For the body weight change detection result of the snakehead after immunization with the engineering bacteria, and the local enlarged view is the body weight change detection result at 120 h; Figure 11 For the detection result of the number of the engineering bacteria in the intestinal tract; Figure 12 For the detection results of (A) CD80 + CD11C + DCs cell proportion, (B) CD86 + CD11C + DCs number in the intestinal tissue; Figure 13 For the detection results of B220 + IgT + B cell number in the intestinal tissue and the spleen, (A) intestinal tissue, and (B) spleen; Figure 14 For the detection results of CD4 + CD8 + T cell number in the intestinal tissue and the spleen, (A) intestinal tissue, and (B) spleen; Figure 15 For the detection results of CD3 + CD4 + T cell number in the intestinal tissue and the spleen, (A) intestinal tissue, and (B) spleen; Figure 16 For the detection results of the IL-10 gene mRNA transcription level in the intestinal tract of the snakehead, (A) detection result after the first immunization, and (B) detection result after the booster immunization; Figure 17The mRNA transcription level detection results of IL-6 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 18 The mRNA transcription level detection results of IL-1β gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 19 The mRNA transcription level detection results of TNF-α gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 20 The mRNA transcription level detection results of TLR4 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 21 The mRNA transcription level detection results of TLR5 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 22 The mRNA transcription level detection results of MyD88 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 23 The mRNA transcription level detection results of Hsp60 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 24 The mRNA transcription level detection results of IκB-α gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 25 The mRNA transcription level detection results of p53 gene in the intestine of snakehead, (A) detection results after the first immunization; (B) detection results after the booster immunization; Figure 26 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 snakehead and (F) expression detection results; Figure 27 The influence of the engineering bacteria on the protein expression of (A) Occludin, (B) Claudin1, (C) ZO-1 and (D) E-cadherin in the intestine of snakehead and (E) expression detection results; Figure 28 The intestinal flora species composition classification results of snakehead; Figure 29 The intestinal histopathological detection results of snakehead after challenge; In the above figures, ns represents no significant difference, * represents P value < 0.05: ** represents P value < 0.01, *‌** represents P value < 0.001, *‌*** represents P value < 0.0001; Control is a blank control group, A. veronii For A. veronii TH0426, LP wt For L. plantarum NC8, LP-pPG-aha is an engineering bacteria LP-pPG-aha, LP-pPG-Inp is an engineering bacteria LP-pPG-Inp, LP-pPG-CsgB is an engineering bacteria LP-pPG-CsgB, and LP-pPG-CsgF is an engineering bacteria LP-pPG-CsgF. DETAILED DESCRIPTION

[0020] 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.

[0021] The application will be described in further detail below with specific examples. It should be noted that the specific examples are illustrative of the application and are not limiting.

[0022] Sources of all biological materials (genes, vectors, microorganisms, etc.): L. plantarum NCBI Reference Sequence of NC8: NZ_AGRI01000002.1, A. veronii NCBI database information of TH0426: BioProject: PRJNA293940, BioSample: SAMN04012505, Lactobacillus casei anchor expression vector pPG612 plasmid purchased from BNCC Company; pEASYBlunt cloning vector purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0023] Example 1: Amplification of aha, Inp, CsgB, CsgF genes and construction of recombinant plasmid The construction method is as shown in Figure 1 Specifically includes: 1. Acquisition and amplification of target genes: The strain A. veronii TH0426 was activated in LB broth at 37°C for 8h, and after three generations, the LB agar plate was streaked and cultured at 37°C for 12h. Single colonies were picked and purified. The purified bacterial solution was used to extract the genome.

[0024] According to the GenBank registration A. veronii The whole genome data (CP012504.1) of TH0426 was used to obtain the gene sequences of aha, Inp, CsgB and CsgF (SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4), and four pairs of specific primers for connecting the lactobacillus 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, Inp gene fragment (F2, R2) containing Nco I, Sma I restriction site, CsgB gene fragment (F3, R3) containing Nco I, Not I restriction site, and CsgF gene fragment (F4, R4) containing Nco I, Sac II restriction site. The primers were synthesized by Chunxi Library Company in Changchun.

[0025] The expected fragment sizes of the primers are 1038 bp for aha gene, 381 bp for Inp gene, 1590 bp for CsgB gene, and 840 bp for CsgF gene. The extracted A. veronii TH0426 genome was used as the template for amplification of the four pairs of primers. The amplification conditions were as follows: pre-denaturation (95℃, 5min); denaturation (94℃, 1min), annealing (aha: 53℃, Inp: 68℃, CsgB: 68℃, CsgF: 68℃, 1min), extension (72℃, 1min) for 30 cycles; and further extension (72℃, 10min). After PCR amplification, 5μL of the product was taken for electrophoresis detection, and the results were observed by gel imaging instrument. The target genes were recovered using OMEGA gel recovery kit and stored at -20℃.

[0026] The PCR reaction system was as follows: PrimeSTAR Max 10μL, upstream primer 1μL, downstream primer 1μL, template 1μL, ddH2O (double distilled water) 7μL, and the total system was 20μL.

[0027] 2. The frozen strain E. coli MC1061 was used to prepare competent cells; 3. Construction and identification of the cloning vector: The aha, Inp, CsgB and CsgF gene fragments recovered from the glue were respectively connected to pEASY-Blunt cloning vector, and reacted at 25°C for 4h. The connection system was as follows: pEASYBlunt Vector 1 μL, target gene 4 μL, and the reaction system was 5 μL.

[0028] The connection product was transformed into MC1061 competent cells by ordinary transformation method, including: (1) Competent cells E. coli MC1061 was ice-bathed for 15 min, and after recovery, 50 μL was mixed with 5 μL of connection product, and ice-bathed for 30 min; (2) 42°C heat shock for 90 s, ice-bath for 3 min 40 s; (3) Add 800 μL LB broth to 37°C shaker for 2.5 h; (4) After the bacterial solution was turbid, it was centrifuged in a centrifuge at 5000 rpm for 8 min, and the supernatant was discarded. 100 μL of resuspended bacteria were coated on LB agar plates (ampicillin solution 50 μg / mL), and incubated at 37°C for 16 h. Single colony PCR identification was performed, and suspected plasmids were sent for sequence detection. After correct identification, they were named as pEASYBlunt-aha, pEASYBlunt-Inp, pEASYBlunt-CsgB, and pEASYBlunt-CsgF, respectively.

[0029] 4. Expression vector and target gene cloning: The pPG612, pEASYBlunt-aha, pEASYBlunt-Inp, pEASYBlunt-CsgB, and pEASYBlunt-CsgF plasmids were respectively double-digested, and the enzyme digestion reaction system was as follows: template (plasmid) 25 μL, buffer (10×) 5 μL, restriction enzyme (EcoRI) 1.5 μL, restriction enzyme (BamHI) 1.5 μL, and ddH2O 17 μL. EcoR V / Nco I / Sac II) 1.5 μL, restriction enzyme (EcoRI) 1.5 μL, restriction enzyme (BamHI) 1.5 μL, and ddH2O 17 μL. Sma I / Not I) 1.5 μL, ddH2O 17 μL.

[0030] 5. Connection of expression vector and target gene sequence: The pPG612 and target gene were connected by T4 DNA ligase, and the connection product was transformed into E. coliMC1061 competent. The reaction system 16℃ overnight, the bacteria liquid culture 2.5h be coated on LB agar plate (chloramphenicol concentration Cm:10 μg / mL), pick single colony using PCR and enzyme cutting preliminary identification, after the suspected strain sent to test sequence. The strain named pPG-aha, pPG-Inp, pPG-CsgB and pPG-CsgF for sequencing error.

[0031] T4 connection reaction system is T4 DNA ligase (ligase) 2 μL, T4 DNA ligase buffer (10×) 1 μL, the target gene fragment 5.5 μL, carrier fragment 1.5 μL, the reaction system is 10 μL.

[0032] Example 2: construction and verification of recombinant lactic acid bacteria 1. Preparation L. plantarum NC8 competent: (1) the constructed strain was inoculated in liquid MRS broth, and three generations were inoculated on MRS agar plate, and single colony was picked for purification; (2) the purified bacteria liquid was inoculated into 200 mL MRS broth at a ratio of 2wt%, and cultured at 37℃ under anaerobic condition to OD 600 nm=0.6 (6 h); (3) the cultured bacteria liquid was transferred to a sterile 50 mL centrifuge tube, and centrifuged at 4℃, 4000 rpm for 15 min after ice bath for 30 min, and the supernatant was discarded; (4) the bacterial pellet was resuspended twice with 20 mL pre-cooled EPWB (0.0936g NaH2PO4·2H2O and 0.0213g MgCl2·6H2O, ddH2O to 1 L), and centrifuged at 4℃, 4000 rpm for 15 min, and the supernatant was discarded; (5) the bacterial pellet was collected and resuspended with 20 mL pre-cooled EPB (200 mL EPWB and 20.53782g sucrose), and centrifuged at 4℃, 4000 rpm for 15 min, and the supernatant was discarded; (6) the bacterial pellet was resuspended with 1 mL pre-cooled EPB, ice bath for 20 min, and distributed in pre-cooled 1.5 mL centrifuge tube at 100 μL / tube, and stored at-80℃.

[0033] 2. Electroporation: The correct recombinant plasmid was transformed into L. plantarum NC8 competent cells by electroporation, including: (1) the L. plantarum NC8 competent cells were thawed for 15 min, then 200 μL was mixed with the constructed recombinant plasmid (10 μL), and ice bathed for 10 min; (2) Shock cup pre-cooled to 4°C, transfer mixture to shock cup (2 mm), ice bath for 10 min; (3) Shock conditions: 2.52 kV, 5 ms; (4) After the shock, quickly add 900 μL of pre-heated MRS broth (containing 15 wt% sucrose), ice bath for 20 min, then place in a 37°C incubator for anaerobic culture for 3 h; (5) Take 100 μL of turbid bacterial solution and spread on MRS agar plates (Cm: 10 μg / mL) at 37°C for 36 h of anaerobic culture; 3. Recombinant lactic acid bacteria gene level verification The next day, single colonies were picked into 10 mL of MRS broth (Cm: 5 μg / mL) and incubated anaerobically at 37°C overnight; 4 mL of bacterial solution was used to extract plasmids. The plasmids were resuspended with 250 μL of P1 solution from a small plasmid extraction kit, then 250 μL of lysozyme (100 mg / mL) was added, and the wall was broken 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.

[0034] Example 3: Recombinant lactic acid bacteria protein verification 1. Determination of the growth curve of the engineered bacteria; 2. Preparation of recombinant bacterial proteins: The recombinant bacteria were inoculated in MRS liquid medium (Cm: 5 μg / mL), and after 2 generations, they were inoculated in MRS broth (Cm: 5 μg / 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 three times with 2 mL of sterile PBS (phosphate buffered solution), and each time it was centrifuged (4°C, 8000 rpm, 12 min). After resuspension in 1 mL of PBS, 500 μL of lysozyme was added for wall breaking (37°C, 1 h), and after wall breaking, the precipitate was collected by centrifugation (4°C, 8000 rpm, 12 min). Protein quantification was performed using a BCA kit, and sterile PBS was used to dilute it to 10 μg / μL. 400 μL of the resulting protein was mixed with 100 μL of SDS-PAGE buffer (5x), boiled at 100°C for 8 min, and then stored at -80°C after denaturation.

[0035] 3. SDS-PAGE: Separation gel and concentration gel were prepared according to the preparation box of Beijing Sunshine Gel.

[0036] 4. Western blot and indirect immunofluorescence.

[0037] Example 4: Biological characteristics analysis of Hsp60 receptor-targeting engineered bacteria 1. AB.9 cell resuscitation, medium change, passage, and cryopreservation; 2. Bacterial culture: (1) A. veronii Preparation of TH0426 bacterial culture a. Selection A. veronii The 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. b. Adjust the concentration to 1.5 × 10⁻⁶ using complete culture medium. 7 CFU / mL, to be used during cell incubation.

[0038] (2) Preparation of engineered bacteria culture 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; b. Adjust the concentration to 1.5 × 10⁻⁶ using complete culture medium. 7 CFU / mL, to be used after incubation with cells.

[0039] 3. Detection of the optimal interaction time between bacteria and AB.9 cells: 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.

[0040] 4. Recombinant lactic acid bacteria and A. veronii Assay of TH0426's ability to adhere to AB.9 cells: (1) The four engineered strains and the wild strain were constructed. L. plantarum NC8 and A. veroniiTH0426 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; (2) Take the plateau phase bacterial culture, wash three times with pre-cooled PBS, pre-cool to 4°C, centrifuge at 5000 rpm for 10 min, collect the bacterial cells, and resuspend in MEM incomplete culture medium. Inoculate the bacterial culture into monolayer cells of 6-well plates at an infection multiple (MOI) of 100:1 (1.5 × 10⁻⁶ cells / well). 7 1.5 × 10⁶ bacteria 5 (Cellular action) (3) After culturing in a 25℃, 5% CO2 incubator for 6 hours ( A. veronii After culturing for 4 hours, discard the complete culture medium and wash the cells three times with pre-warmed PBS to remove any unattached cells. Be gentle during washing to avoid detaching the cells. (4) After washing, add 200 μL of 1% Triton X-100 to each well and incubate at 25°C for 30 min. Dilute the lysate with PBS at a ratio of 1:10 and spread it on MRS solid medium (MRS medium for recombinant bacteria contains Cm: 10 μg / mL). Count the number of adhering bacteria. Incubate anaerobically overnight at 37°C. Perform three replicates per group to reduce error. Adhesion rate = number of adhering bacteria / total number of bacteria × 100%.

[0041] 5. Engineered bacteria against A. veronii Competitive adhesion ability test: (1) Four recombinant lactic acid bacteria and A. veronii TH0426 was adjusted to 1.5 × 10⁻⁶ using incomplete culture medium. 7 CFU / mL; (2) Using four recombinant lactic acid bacteria and A. veronii TH0426 was co-infected with monolayer AB.9 cells for 4 h (25℃, 5% CO2 culture). Control was the blank control group, and the added reagent was PBS buffer. (3) Wash the cells three times with pre-warmed PBS to remove any unattached bacterial cells. After washing, add 200 μL of 1% Triton X-100 to each well and incubate at 25°C for 30 min. This process is repeated for four types of recombinant lactic acid bacteria. A. veronii TH0426 was diluted with PBS and counted using a droplet plate. The adhesion rate and adhesion reduction rate were calculated separately. Competitive adhesion rate = (Number of competing bacteria / Total number of bacteria) × 100%; Adhesion reduction rate = Adhesion rate - Competitive adhesion rate.

[0042] 6. Recombinant lactic acid bacteria and A. veronii Assay of TH0426's ability to invade AB.9 cells: (1) Preparation A. veronii TH0426 bacteria solution and four kinds of recombinant lactic acid bacteria suspension; (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; (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; (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%.

[0043] 7. Competition invasion ability detection of the engineering bacteria A. veronii Four kinds of recombinant lactic acid bacteria and A. veronii TH0426 were used to co-infect the monolayer cells for 4 h, and after the cells were washed three times with pre-warmed PBS, 200 μL of incomplete medium containing gentamicin was added to each well 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.

[0044] 8. Detection of the mRNA expression level of the AB.9 cell-related factor of the engineering bacteria

[0045] Example 5: Evaluation of the immune effect of the Hsp60 receptor-targeted engineering bacteria 1. Immunization scheme of the test animals: (1) The Trichogaster trichopterus 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.

[0046] Table 1 Grouping of the test animals

[0047] ​(2) Examine and collect feces and intestinal contents. Use MS-222 reagent (250mg / L, Sigma, USA) to anesthetize and kill snakehead fish in each group. Disinfect the fish surface with 75% alcohol to remove bacteria. After dissection, collect snakehead intestinal tissue and wash off the attached blood with sterile PBS. After quick freezing in liquid nitrogen, store at -80°C.

[0048] (3) Flow cytometry was used to detect engineered bacteria.

[0049] 2. Preparation of cell suspension; 3. DCs detection; 4. B cell detection; 5. T cell detection; 6. Detection of recombinant lactic acid bacteria intestinal colonization: On day 6 after the first oral immunization, gut microbiota colonization was assessed. Testing was conducted every 12 hours from 24 hours to 72 hours post-fasting, for a total of 5 tests, with 3 microbes tested each time. Intestinal tissue was harvested from necropsy, flash-frozen in liquid nitrogen, and rapidly homogenized. The homogenized tissue was then transferred to sterile centrifuge tubes (containing 1 mL of sterile PBS buffer), serially diluted, and counted using platelets. Three replicates were set up, and colonies were randomly selected for PCR validation.

[0050] 7. Detect the expression levels of cytokine mRNA; 8. Detection of the effect of engineered bacteria on the expression of gut-associated proteins in snakehead fish; 9. After the virus challenge, intestinal and spleen tissues were collected from each group of snakehead fish for histopathological examination; 10.16S Microbial Diversity Detection: Fresh feces and intestinal contents of snakehead fish from each group were collected 18 days after immunization to detect microbial diversity. The species composition was determined by sequence detection and intestinal microbial diversity was analyzed by Alpha and Beta diversity analysis on the Pasenno Gene Cloud Platform.

[0051] Experimental results show that: like Figure 2 As shown, the growth rate of the engineered strain was basically the same as that of the wild-type strain, with no significant difference, indicating that the expression of the target protein had no significant effect on the growth of the host strain. Figure 3 As shown, this demonstrates that the protein in L. plantarum It can be stably expressed in NC8; such as Figure 4 As shown, compared to L. plantarum NC8, engineered bacteria have superior growth performance, such as Figure 5 As shown, compared with the control group, A. veronii When the treatment time was 5 h, the cell viability was significantly lower than that of the control group (P<0.01). Figure 6The results show the effect of engineered bacteria on the survival rate of AB.9 cells; (A) 2h, (B) 4h, (C) 6h, (D) 8h; compared with the blank control group, L. plantarum When the treatment time for NC8 and engineered bacteria was 8 h, the cell survival rate was significantly lower than that of the control group.

[0052] like Figure 7 As shown in Figures (A, B, C), compared with other strains, the engineered strains LP-pPG-CsgB and LP-pPG-CsgF exhibit superior adhesion and competitive adhesion abilities. A. veronii The rate of decrease in cell adhesion of AB.9 was significantly higher than that of engineered bacteria.

[0053] like Figure 7 As shown in (D, E, F), the engineered bacterium LP-pPG-CsgF exhibits superior invasiveness and competitive invasion ability compared to other strains. A. veronii , A. veronii The rate of decrease in invasion was significantly higher than that of engineered bacteria.

[0054] Figure 8 To detect the expression levels of cell-related factors mRNA in AB.9 cells by engineered bacteria, (A) results of Hsp60 gene mRNA level detection in AB.9 cells, (B) results of IL-10 gene mRNA level detection in AB.9 cells, (C) results of IL-6 gene mRNA level detection in AB.9 cells, (D) results of IL-β gene mRNA level detection in AB.9 cells, and (E) results of TNF-α gene mRNA level detection in AB.9 cells; the expression levels of Hsp60 and IL-10 gene in the engineered bacteria LP-pPG-CsgB and LP-pPG-CsgF groups were significantly higher than those in the blank control group, while the expression levels of IL-1β, IL-6, and TNF-α genes in the engineered bacteria LP-pPG-CsgB and LP-pPG-CsgF groups were lower than those in the blank control group.

[0055] 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. 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; Figure 11 Results of the detection of the number of engineered bacteria colonizing the gut; Figure 12CD80 + CD11C + CD86 + CD11C + The results of detecting the number of DCs, the engineered probiotics LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF can increase the number of CD80 + cells and CD86 + cells, indicating that the constructed targeted engineered bacteria can promote the differentiation and maturation of dendritic cells (DCs), and further promote the immune system to clear the intestinal pathogens. Figure 13 The results of detecting the number of B220 + IgT + cells in the intestinal tissue and the spleen, (A) intestinal tissue; (B) spleen; Figure 14 The results of detecting the number of CD4 + CD8 + cells in the intestinal tissue and the spleen, (A) intestinal tissue; (B) spleen; Figure 15 The results of detecting the number of CD3 + CD4 + cells in the intestinal tissue and the spleen, (A) intestinal tissue; (B) spleen; LP-pPG-aha, LP-pPG-Inp, LP-pPG-CsgB and LP-pPG-CsgF can stimulate the proliferation of immune-related cells in the intestinal tract and the spleen. It is shown that the oral engineered bacteria can be taken up by DC cells, treated, and stimulate T cells and B cells to play cellular immunity and humoral immunity, which lays a foundation for the development of probiotic oral vaccines.

[0056] Figure 16 The results of detecting the mRNA transcription level of IL-10 gene in the intestinal tract of snakehead, (A) the results after the first immunization; (B) the results after the booster immunization; Figure 17 The results of detecting the mRNA transcription level of IL-6 gene in the intestinal tract of snakehead, (A) the results after the first immunization; (B) the results after the booster immunization; Figure 18 The results of detecting the mRNA transcription level of IL-1β gene in the intestinal tract of snakehead, (A) the results after the first immunization; (B) the results after the booster immunization; Figure 19 The results of detecting the mRNA transcription level of TNF-α gene in the intestinal tract of snakehead, (A) the results after the first immunization; (B) the results after the booster immunization; Figure 20 The results of detecting the mRNA transcription level of TLR4 gene in the intestinal tract of snakehead, (A) the results after the first immunization; (B) the results after the booster immunization; Figure 21is the detection result of the mRNA transcription level of TLR5 gene in the intestine of Channa argus, (A) detection result after the first immunization; (B) detection result after the booster immunization; Figure 22 is the detection result of the mRNA transcription level of MyD88 gene in the intestine of Channa argus, (A) detection result after the first immunization; (B) detection result after the booster immunization; Figure 23 is the detection result of the mRNA transcription level of Hsp60 gene in the intestine of Channa argus, (A) detection result after the first immunization; (B) detection result after the booster immunization; Figure 24 is the detection result of the mRNA transcription level of IκB-α gene in the intestine of Channa argus, (A) detection result after the first immunization; (B) detection result after the booster immunization; Figure 25 is the detection result of the mRNA transcription level of p53 gene in the intestine of Channa argus, (A) detection result after the first immunization; (B) detection result after the booster immunization; Figure 26 is 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 Channa argus and (F) expression detection result; Figure 27 is the influence of the engineering bacteria on the protein expression of (A) Occludin, (B) Claudin1, (C) ZO-1 and (D) E-cadherin in the intestine of Channa argus and (E) expression detection result; 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 Channa argus, indicating that the adhesion proteins expressed by the engineering bacteria can specifically bind to the Hsp60 receptor of Channa argus 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. The 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. It is found in the present application that after oral immunization of the engineering bacteria, the anti-inflammatory factors of Channa argus in each group continue to increase, 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 Channa argus, and the bacterial flora can mediate and activate the immune response through the feeding environment, resist A. veronii infection, and provide an effective treatment approach for inflammatory bowel disease and other gastrointestinal diseases.

[0057] The above merely provides 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 modifications, equivalent replacements, improvements, etc. made within the principles and technical scope of the present application shall fall into the 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.

2. The Hsp60 receptor-targeted recombinant plant lactobacillus of claim 1, wherein, The Lactobacillus plantarum is Lactobacillus plantarum L. plantarum NC8.

3. 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.

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

5. The use of Hsp60 receptor-targeting recombinant Lactobacillus plantarum in the production of proteins according to claim 1 or 2.

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

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

8. Use of the Hsp60 receptor-targeted recombinant plantarum lactis bacterium according to claim 5 for the production of proteins, characterized by the fact that, comprising: The Hsp60 receptor-targeting recombinant Lactobacillus plantarum according to claim 1 or 2 is inoculated into MRS broth medium to ferment and produce proteins.

9. The use of Hsp60 receptor-targeting recombinant Lactobacillus plantarum according to claim 1 or 2 in the preparation of oral vaccine for preventing and treating Veillonella wautersi.

10. The use of Hsp60 receptor-targeting recombinant Lactobacillus plantarum according to claim 1 or 2 in the preparation of drugs for treating inflammatory bowel disease.

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