Gfp surface visualized labeling based detection-feedback regulation method for plantaricin colonization in paralichthys olivaceus and its application

Abstract

The application discloses a detection-feedback regulation method for plant lactobacillus colonization based on GFP surface visualization marking and application, and belongs to the field of aquaculture and probiotic application.The method comprises the following steps: constructing a recombinant shuttle vector pGLP, using the recombinant shuttle vector pGLP to construct a recombinant strain LP-GFP, using the recombinant strain LP-GFP to prepare a feed for paralichthys olivaceus with a fluorescent tracing effect, carrying out initial feeding, carrying out stage sampling detection, analyzing the time-space colonization rule of LP-GFP in the foregut-midgut-hindgut of paralichthys olivaceus, obtaining the most important target position, and dynamically regulating the feeding strategy according to the detection result.The application establishes a closed loop of 'detection-feedback-dynamic regulation', breaks through the traditional fixed time length and fixed dose experience feeding mode, uses the fluorescent colonization detection as a feedback control signal, avoids the situation that probiotics are ineffective due to insufficient feeding, and also avoids the situation that excessive feeding causes feed waste and intestinal burden, and solves the technical pain points of blindly feeding probiotics for aquatic fish.

Description

Technical Field

[0001] This invention relates to a method and application for colonization detection and feedback regulation of Lactobacillus plantarum derived from turbot based on GFP surface visualization markers, belonging to the fields of aquaculture and probiotic applications. Background Technology

[0002] Turbot is an important marine aquaculture species in northern my country. Under intensive, factory-style farming conditions, high density and fluctuating water quality easily induce bacterial diseases such as Vibrio and Edwardsiella tarda. With the reduction of antibiotic use, probiotics are an important alternative technology for regulating intestinal health and preventing diseases.

[0003] For probiotics to exert their beneficial effects, they must achieve effective and stable adhesion and colonization in the host's gut, rather than merely passing through the digestive tract briefly. Current technologies for evaluating the colonization of aquatic probiotics have significant shortcomings: Traditional antibiotic plate counting and conventional PCR can only obtain the total DNA or viable count of the bacterial community, but cannot determine the spatial location of the strains in the intestine, or distinguish whether the strains are free in the intestinal contents or truly attached to the mucus layer or intestinal epithelium; the counting results cannot explain the reality of "the viable count meets the standard, but the probiotic effect is poor".

[0004] Most of the existing GFP-labeled lactic acid bacteria are exogenous strains from terrestrial origin, which do not match the intestinal physiological environment of turbot and have weak adhesion ability. The labeling results cannot reflect the true interaction patterns of native probiotics. Most GFP is expressed intracellularly, and the fluorescence is easily quenched, making it difficult to observe stably in intestinal tissue sections.

[0005] Existing surface display vectors for lactic acid bacteria are mostly inducible vectors, which require the addition of external inducers and are difficult to implement in the breeding field; some anchoring elements are easily lost during passage of exogenous genes, and the in vivo tracking window is short.

[0006] Current research models involve fixed feeding doses and durations, followed by post-culture testing of colonization and immune indicators. However, there is a lack of technical means to dynamically adjust probiotic feeding strategies using colonization test results as feedback signals. Probiotic feeding dosages and cycles rely entirely on experience: insufficient feeding results in intestinal colonization failing to reach the effective threshold; excessive feeding leads to feed waste and increases the metabolic burden on the turbot's intestines. Furthermore, the development of oral live vector vaccines lacks rapid in vivo evaluation methods for assessing the "colonization efficiency of antigen-delivering strains at the target site," relying solely on inverse estimations based on final immune protection results, resulting in long development cycles.

[0007] Green fluorescent protein (GFP) requires no substrate and enables real-time fluorescence observation of live bacteria. This invention uses the highly adhesive native Lactobacillus Plantarum PO23 (LP-PO23) isolated from the intestines of turbot as the starting strain, and constructs a constitutive GFP surface displaying recombinant LP-GFP using LPTTG cell wall anchoring motifs. This invention not only uses fluorescence colonization detection as an endpoint verification method, but also establishes a closed-loop technical system of "strain labeling - colonization visualization detection - adjustment of feeding strategy based on colonization quantitative / localization results - probiotic effect evaluation / vaccine chassis screening." On the one hand, it enables intuitive tracking of the spatial distribution and colonization dynamics of probiotics in the gut; on the other hand, it utilizes the quantitative and localization data from colonization detection to solve the technical pain points of blindly feeding probiotics in aquaculture and the difficulty in quickly assessing the in vivo colonization efficiency of live vector vaccines.

[0008] Currently, there is a lack of native probiotic tracer strains specifically for turbot. Therefore, it is necessary to explore a method for detecting colonization and regulating aquaculture based on feedback from turbot. Summary of the Invention

[0009] The purpose of this invention is to provide a method and application for colonization detection and feedback regulation of Lactobacillus plantarum derived from turbot based on GFP surface visualization markers, so as to overcome the shortcomings of the prior art.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows: A feedback regulation method for colonization detection of Lactobacillus plantarum derived from turbot based on GFP surface visualization markers includes the following steps: 1) Construction of the recombinant shuttle vector pGLP Using pMG36e as the backbone, the GFP gene was fused with the LPXTG anchoring motif derived from Lactobacillus plantarum lp_2578 to construct the recombinant shuttle vector pGLP.

[0011] 2) Construct the recombinant strain LP-GFP using the recombinant shuttle vector pGLP obtained in step 1). pGLP was electroporated into wild-type turbot-derived Lactobacillus plantarum LP-PO23, and recombinant strain LP-GFP was obtained by screening. The strain was continuously passaged in vitro to screen for engineered strains with stable plasmids. Only strains that passed the passage stability verification could be used for feed preparation.

[0012] Its characteristic is that it also includes the following steps: 3) Using the recombinant strain LP-GFP constructed in step 2), prepare turbot tracer-probiotic feed with fluorescent tracer effect; The prepared LP-GFP was cultured and passaged for more than 20 generations to obtain a stable strain; the stable LP-GFP cells were collected by centrifugation and resuspended in buffer. The bacterial solution was uniformly mixed into commercial turbot feed powder and pelleted feed was prepared; the pellets were then aseptically and slowly air-dried to obtain turbot tracer-tracer probiotic feed.

[0013] 4) Perform initial feeding Initial feeding stage: Turbot should be fed the above-mentioned tracer-probiotic feed at a daily feeding rate of 1% of their body weight; feeding should be done at 8:00 am every day; the water should be changed 1 / 5 of the way after feeding, and the uneaten feed and feces at the bottom of the pond should be removed.

[0014] 5) Periodic sampling and testing: After 7 days of continuous feeding, samples of experimental turbot were randomly selected for testing; the testing included three dimensions: ① Colonization count detection: The foregut, midgut and hindgut tissues of turbot were separated, the contents of the intestinal lumen were removed, the intestinal lumen was washed with sterile PBS to remove free bacteria, the tissues were ground, and the number of LP-GFP live bacteria adhering to the intestinal tissues was obtained by counting on erythromycin-resistant MRS plates. ② Colonization spatial localization detection: Frozen sections of intestinal tissue were prepared and observed under a fluorescence microscope to determine whether LP-GFP was effectively colonized at the mucus layer-intestinal epithelial interface (distinguishing between free intestinal lumen and true adhesion colonization). ③Intestinal physiological and immune detection: Hematoxylin and eosin (HE) staining of paraffin sections was used to observe the morphology of intestinal villi and mucosa; qRT-PCR was used to detect the expression levels of intestinal IL-1β, TNF-α, IL-10, and occludin genes.

[0015] 6) Analyze the temporal and spatial colonization patterns of LP-GFP in the foregut-midgut-hingut of turbot to obtain the most important localized target sites; 7) Dynamically adjust the feeding strategy based on the test results. Set a live bacteria adhesion threshold. When the live bacteria adhesion in the intestinal tissue is greater than or equal to the live bacteria adhesion threshold, and LP-GFP is observed to be distributed at the mucus-epithelial interface in fluorescent sections, it indicates that effective colonization has been achieved. Stop feeding turbot tracers-probiotic feed and switch to ordinary commercial feed. Rely on the LP-GFP that has been colonized in the intestine to continue to exert its probiotic effect. Subsequently, samples can be taken at fixed intervals to monitor the colonization decline. When the colonization amount falls below the threshold, feeding can be restarted. When the number of live bacteria adhering to the intestinal tract is less than the live bacteria adhesion threshold, the fluorescence signal is weak and effective colonization has not been achieved: continue to feed the tracer-probiotic feed, and repeat the sampling and testing every 3-5 days until the effective colonization threshold is reached.

[0016] 8) Follow-up evaluation of probiotic effects After completing the strategy adjustment, we continuously tracked intestinal morphology, tight junction proteins, and cytokine expression to evaluate the effects of immune balance and intestinal barrier improvement.

[0017] In step 3), the final concentration of LP-GFP is adjusted to 1×10⁻⁶ by adjusting the bacterial culture concentration. 9 CFU / g was used to obtain turbot tracer-probiotic feed; after preparation, it was stored at -20℃ in a sealed container away from light; before each use, the actual number of live bacteria in the feed was checked by plate counting; if the loss of live bacteria was >80%, the feed was prepared again.

[0018] Step 6) Obtaining the most important fixed target sites is as follows: The temporal-spatial colonization pattern is obtained by quantitative counting and fluorescent section localization at multiple time points (1, 3, 7, 14, and 21 dpcf after feeding cessation).

[0019] The multiple time points mentioned in step 6) are 1, 3, 7, 14, and 21 days after feeding was stopped.

[0020] The temporal-spatial colonization pattern is as follows: colonization abundance: midgut > foregut > hindgut; the midgut is the most important colonization target site for LP-GFP. After feeding is stopped, the viable bacteria gradually decrease over time, and a small number of adhering viable bacteria can still be detected after 21 days.

[0021] In step 7), the number of live bacteria adhering to the midgut tissue is ≥10. 4 CFU / g tissue represents the adhesion threshold of live bacteria.

[0022] Obviously, the above method can achieve precise feeding of feed containing probiotics. Therefore, one application of the detection-feedback control method is the precise feeding of probiotics in the industrialized farming of turbot.

[0023] This method can also be used to evaluate the effects of aquaculture stress (high temperature, high density) on the colonization ability of native probiotics.

[0024] This method can also be used to assess the feeding time required to reach the effective colonization threshold under different aquaculture environments. Beneficial effects

[0025] 1) LP-PO23 was isolated from the native gut of turbot. Compared with exogenous lactic acid bacteria, it has a stronger ability to adhere to and colonize the gut, and the tracing results are more consistent with the actual behavior of cultured probiotics. 2) The native Lactobacillus plantarum LP-PO23 from turbot is used to achieve covalent display of GFP on the cell wall surface by combining LPTTG anchoring; the constitutive pMG36e vector does not require an inducer; the plasmid is stable after 20 generations of continuous passage; the growth performance is close to that of wild-type strains; unlike intracellular GFP, the fluorescence localization in tissue sections is clear, which can distinguish whether the strain is free in the intestinal lumen or adheres to the intestinal mucus-epithelial interface; avoiding the defect of simple plate counting that cannot distinguish between "transient bacteria" and true colonizing bacteria.

[0026] 3) Methodological Innovation: This invention establishes a closed-loop system of "detection-feedback-dynamic control," breaking through the traditional fixed-duration, fixed-dosage empirical feeding model. Existing technologies all use fixed feeding days, neglecting individual / batch differences in colonization efficiency caused by the aquaculture environment and fish condition. This invention uses fluorescence colonization detection as a feedback control signal, stopping feeding only when the detection reaches the effective midgut colonization threshold; avoiding insufficient feeding leading to ineffective probiotics, and also avoiding overfeeding causing feed waste and intestinal burden; thus solving the technical pain point of blindly feeding probiotics to aquaculture fish. Attached Figure Description

[0027] Figure 1 This is a flowchart of the overall process of the colonization detection-feedback regulation closed-loop technology of GFP-labeled recombinant Lactobacillus plantarum in this invention.

[0028] Figure 2 Maps of plasmid pMG36e and recombinant pGLP vector, where A. pMG36e plasmid; B. pMG36e-GFP-LPXTG plasmid.

[0029] Figure 3 Fluorescence detection images of recombinant Escherichia coli strains, where A represents E. coli successfully transformed into the pMG36e-GFP-LPXTG plasmid under bright field microscopy, B represents E. coli successfully transformed into the pMG36e-GFP-LPXTG plasmid under the fluorescence channel of a microscope, C represents E. coli successfully transformed into the pMG36e empty plasmid under bright field microscopy, and D represents E. coli successfully transformed into the pMG36e empty plasmid under the fluorescence channel of a microscope.

[0030] Figure 4 Fluorescence micrographs of recombinant Lactobacillus plantarum, where A is LP-GFP under bright field of a microscope; B is LP-GFP under the fluorescence channel of a microscope; C is a combined image of A and B; A1 is LP-36e under bright field of a microscope; B1 is LP-36e under the fluorescence channel of a microscope; C1 is a combined image of A1 and B1.

[0031] Figure 5 To verify GFP expression using SDS-PAGE and Western blotting.

[0032] Figure 6Immunoelectron microscopy was used to observe the localization of colloidal gold LP-36e in the cell wall, where A1 is a cross-sectional view of LP-36e; A2 is a longitudinal view of LP-36e.

[0033] Figure 7 Immunoelectron microscopy was used to observe the colloidal gold localization of LP-GFP in the cell wall, where B1 is a cross-sectional view of LP-GFP and B2 is a longitudinal view of LP-GFP.

[0034] Figure 8 The results are for PCR stability testing of plasmids from 20 generations of passages, where A is the length of the pGLP plasmid and B is the length of the pMG36e plasmid.

[0035] Figure 9 Comparison of growth curves between wild-type and recombinant plants.

[0036] Figure 10 Quantitative analysis of live bacteria colonization at different time points in the foregut, midgut, and hindgut.

[0037] Figure 11 Fluorescent colonization observation of frozen sections of turbot intestine.

[0038] Figure 12 Intestinal HE morphology sections of the blank group and experimental group, where A is the intestinal tissue morphology of the PBS group and B is the intestinal tissue morphology of the LP-GFP group.

[0039] Figure 13 Bar chart showing the relative expression levels of the gut immune-related gene IL-1β.

[0040] Figure 14 Bar chart showing the relative expression levels of the gut immune-related gene TNFα.

[0041] Figure 15 Bar chart showing the expression levels of the gut immune-related gene IL-10.

[0042] Figure 16 Bar chart showing the relative expression levels of the gut immune-related gene Occulin. Detailed Implementation

[0043] This invention provides a closed-loop method for detecting and regulating probiotic colonization in turbot. The method pre-sets an effective intestinal colonization threshold, uses intestinal fluorescence visualization and quantitative detection results as feedback input, and compares the measured colonization indicators with the preset threshold. Based on the comparison results, the probiotic feeding duration and dosage are dynamically adjusted, overcoming the limitations of existing technologies that use fixed duration and dosage based on experience. When the detection results do not reach the preset effective colonization threshold, probiotic feed is continued, and samples are taken again after a specified interval for testing. This testing-judgment process is repeated until the colonization threshold is reached. When the detection results reach the effective colonization threshold, probiotic feed feeding is stopped, and a regular feed is switched to, with periodic colonization monitoring conducted.

[0044] The process of this invention is as follows Figure 1 As shown, the process is as follows: in vitro construction and functional verification of recombinant strains, preparation of tracer-probiotic feed, initial feeding of turbot, and phased multi-dimensional joint sampling and detection. If the effective colonization threshold is reached, the probiotic feed is stopped and the feed is switched to ordinary feed. If the colonization threshold is not reached, the tracer-probiotic feed is continued to be fed, and the sampling and detection process is repeated after a specified interval, forming a closed-loop feedback control. The multi-dimensional joint detection includes quantitative counting of tissue-adherent viable bacteria, spatial localization of fluorescence in frozen sections, observation of intestinal tissue morphology, and quantitative detection of immune genes.

[0045] This invention relates to a method for detecting and regulating colonization of *Lactobacillus tumefaciens* derived from turbot based on GFP surface visualization markers, as detailed below: 1. Construction of the recombinant shuttle vector pGLP Using the constitutive shuttle plasmid pMG36e of lactic acid bacteria as a backbone, a GFP-encoding gene and the LPXTG pentapeptide anchoring sequence from *Lactobacillus plantarum* lp_2578 were fused together, and a GFP-LPXTG fusion expression fragment was inserted to form the fusion expression plasmid GFP-LPXTG. The recombinant plasmid pGLP was then inserted into the vector using Xba I and Hind III double restriction sites to obtain the recombinant plasmid pGLP (e.g., ...). Figure 2 (As shown). The LPTSG sequence is derived from the *Lactobacillus plantarum* lp_2578 gene. The gene involved has been published in the paper "Development and Application of Live Vector Vaccine of *Lactobacillus* with Surface Display of Protective Antigen FlaB of *Vibrio anguillarum*", dated June 2023.

[0046] 2. Construction method of recombinant strain LP-GFP (1) Preparation of competent cells Wild-type LP-PO23 cells were inoculated into MRS medium containing 10 g / L glycine and 0.5 M sucrose and cultured at 37 °C to the logarithmic growth phase. Competent cells were prepared by washing with 30% PEG1500 buffer. The LP-PO23 cells were published in the paper "Isolation, Identification and Adhesion and Colonization Mechanism of Lactobacillus Intrinsic in the Intestine of Turbot" in June 2021. (2) Electroconversion 1 μg pGLP plasmid was mixed with 40 μL competent cells and subjected to a 1.5 kV, 5 ms pulse electric shock in a 2 mm shock cup; (3) Resuscitation screening After 2 h of recovery in MRSSM medium, the bacteria were plated on erythromycin-resistant MRS plates and positive strains LP-GFP were screened to obtain a GFP-displaying recombinant Lactobacillus plantarum LP-GFP.

[0048] 3. Functional validation system for recombinant strains (1) In vitro validation SDS-PAGE and WB were used to verify GFP expression; fluorescence microscopy and immunoelectron microscopy were used to verify protein localization on the bacterial surface; plasmid stability was verified through 20 consecutive passages; growth curves showed that the marker did not significantly inhibit bacterial proliferation; only strains that passed the passage stability verification were allowed to be used for feed preparation and in vivo experiments.

[0049] (2) Closed-loop in vivo colonization tracing and feeding regulation LP-GFP cells were collected, resuspended in buffer, and uniformly mixed into commercial feed powder to prepare pelleted feed. The pellets were then aseptically air-dried at low temperature, with a final viable count of 1×10⁻⁶. 9 CFU / g; store at -20℃, and verify viable counts by plate count before use.

[0050] The study grouped the animals into a control group (feed mixed with an equal volume of PBS) and an experimental group; the animals were initially fed continuously for 7 days; after 7 days, random samples were taken to test the following three dimensions: ① The intestinal tissue was cleaned of intestinal contents, thoroughly rinsed with sterile PBS to remove free bacteria in the intestinal lumen; after grinding, the intestinal tissue was counted on resistant plates to count the live bacteria adhering to the intestinal tissue; ② Observe the fluorescence of frozen sections to confirm whether LP-GFP is localized at the mucus-epithelial interface; ③ HE staining and qPCR detection of intestinal morphology and immune genes.

[0051] Implementation strategy based on test results: If the number of live bacteria adhering to the midgut tissue is ≥10 4CFU / g tissue sections showed GFP fluorescence distribution at the mucus-epithelial interface: indicating that the effective colonization threshold had been reached, and the feed was switched to regular feed; subsequently, colonization was monitored every 7-14 days, and when the colonization decreased below the threshold, probiotic feed was restarted.

[0052] If the number of viable bacteria adhering to the midgut is <10 4 CFU / g tissue, weak fluorescence signal: continue feeding probiotic feed, take samples for retesting every 3-5 days until the colonization threshold is reached.

[0053] Samples were taken at 1, 3, 7, 14 and 21 days after feeding was stopped to analyze the temporal-spatial colonization pattern (midgut > foregut > hindgut, with gradual decrease over time).

[0054] HE staining was used to observe the morphology of intestinal villi; qRT-PCR was used to detect the expression of IL-1β, TNFα, IL-10 and occludin genes, confirming that the strain balanced intestinal immunity and strengthened the intestinal barrier.

[0055] 4. Application scheme of recombinant strains (1) Precise spatial localization and quantification of LP-GFP in the gut of turbot; (2) A method for precise feeding of probiotics to turbot based on colonization detection feedback; (3) As a feed additive, it improves the intestinal morphology of turbot and regulates mucosal immunity; 5. Implementation parameters 1) Electroconversion buffer: 30% (w / v) PEG 1500 aqueous solution; 2) Electric shock parameters: voltage 1.5 kV, pulse duration 5 ms; 3) Feed preparation: The bacterial resuspension buffer is mixed with feed powder and pelleted feed is prepared; the target final viable bacteria concentration is 1×10⁻⁶. 9 CFU / g; Verify viable cell count using plate counting before feeding; 4) The initial feeding period is 7 days, and then the feeding time is dynamically adjusted according to the results of the colonization test; 5) Effective colonization threshold: The effective colonization threshold is when the number of viable midgut bacteria reaches 10³-10⁻¹⁰. 4 CFU / g tissue range, bacterial cell distribution at the mucin-epithelial interface was observed in fluorescent sections; 6) Upper limit of strain stability through passage: ≥20 generations.

[0056] Example 1 Construction of recombinant plasmid pGLP 1. Primers were synthesized to create GFP-F / R and LPXTG-F / R, which were then used to amplify the GFP and lp_2578 anchored fragments, respectively; 2. The two products were recovered, and the GFP-LPXTG fusion fragment was obtained by PCR; 3. The XbaI and HindIII double-digested fusion fragment was ligated into the pMG36e empty vector and incubated overnight at 16 °C with T4 ligase; 4. Conversion of the ligation product into MC1061 (e.g.) Figure 3 As shown in the figure, positive clones were selected for resistance screening, and the sequences were verified to be correct by sequencing. The plasmid pGLP was then extracted for later use.

[0057] Example 2: Electroporation preparation of recombinant strain LP-GFP 1) Activate LP-PO23 for two generations, then inoculate 1:50 into MRS medium containing glycine and sucrose, and incubate at 37°C in a shaker until... ; 2) Collect bacterial cells by centrifugation at 6000 g for 5 min at 4 ℃, and wash 3 times with pre-cooled PEG buffer; 3) Mix 40 μL of competent cells with 1 μg of pGLP plasmid and transfer to an electroporation cuvette; 4) 1.5 kV electric shock, immediately add MRSSM resuscitation solution, and let stand at 37 ℃ for 2 h; 5) Spread erythromycin-containing MRS plates, pick single colonies for culture, and use fluorescence to screen for positive strains such as LP-GFP (e.g., ...). Figure 4 (As shown).

[0058] 6) Western spectroscopy was used to verify that the recombinant protein GFP-LPXTG was successfully expressed in recombinant strains MC1061-GFP and LP-GFP (e.g., Figure 5 (As shown).

[0059] 7) Immunoelectron microscopy revealed that colloidal gold particles were distributed on the surface of LP-GFP cells, and were mainly concentrated on the cell wall surface of LP-GFP cells (e.g., ...). Figure 6 , 7 (As shown).

[0060] Example 3: Stability and growth performance testing of the strain LP-GFP was continuously passaged for 20 generations, and plasmids were extracted and PCR was performed every 5 generations, which amplified the target band (e.g., Figure 8 As shown); the 24-hour growth curve shows that the recombinant strain grows slightly slower only in the logarithmic phase, and the bacterial density in the stationary phase is not significantly different from that of the wild-type strain, and does not affect its in vivo colonization ability (e.g. Figure 9 (As shown).

[0061] Example 4: Visual Tracing Application of Turbot Intestinal Colonization 120 healthy turbot were randomly divided into a control group and an experimental group. The experimental group was fed LP-GFP tracer feed for 7 consecutive days initially, and samples were taken for testing. In some individuals in the experimental group, the number of viable gut-adherent bacteria reached 1.2 × 10⁻⁶. 5CFU / g tissue (e.g.) Figure 10 As shown), frozen sections revealed GFP-positive signals distributed in the intestinal mucus layer near the epithelium, reaching the effective colonization threshold (e.g. Figure 11 (As shown in the figure); stop feeding probiotic feed and switch to ordinary feed; samples were taken at 1, 7, 14 and 21 days after stopping feeding to obtain the colonization dynamics of LP-GFP in the foregut, midgut and hindgut of turbot over time.

[0062] During the experiment, it was found that when subjected to environmental stresses such as water temperature, the number of live gut bacteria in the sample taken after 7 days was only 10² CFU / g tissue, which did not reach the threshold. Therefore, the probiotic feed was continued to be fed, and the colonization threshold was reached when the sample was taken on the 12th day, at which point feeding was stopped.

[0063] This example demonstrates that a fixed 7 / 14-day feeding schedule is not suitable for all breeding batches; by relying on feedback from planting and testing, the feeding cycle can be flexibly controlled for different breeding batches.

[0064] Example 5: Application of recombinant bacterial strains as a probiotic feed additive in aquaculture Using the feedback control strategy of Example 4, feeding was stopped when the colonization threshold was reached; HE staining showed that the intestinal villi in the experimental group were longer and the mucosal layer was thickened (e.g., Figure 12 (As shown); qPCR detection revealed that early pro-inflammatory factors were upregulated, followed by significant upregulation of anti-inflammatory factors IL-10 and tight junction occludin, improving gut health in turbot under high-density farming conditions (e.g. Figures 13-16 (As shown).

[0065] Example 6: Application of recombinant strains in enhancing the adhesion and colonization ability of aquatic probiotics The protective antigen gene of Vibrio anguillarum was inserted into the pGLP vector to construct a batch of candidate recombinant Lactobacillus plantarum. Candidate strains were obtained by electroporation and fed to turbot. The colonization detection method of this invention was used to quickly detect the number of candidate strains colonizing the midgut mucus-epithelial interface of turbot. The midgut colonization ability was used as the initial screening index to eliminate candidate strains with in vivo colonization defects, and only strains with qualified colonization were retained for subsequent challenge protection experiments, reducing the use of experimental animals and shortening the vaccine development cycle.

Claims

1. A feedback regulation method for colonization detection of Lactobacillus plantarum derived from turbot based on GFP surface visualization markers, comprising the following steps: 1) Construct the recombinant shuttle vector pGLP; 2) Construct the recombinant strain LP-GFP using the recombinant shuttle vector pGLP obtained in step 1); Its characteristic is that it also includes the following steps: 3) Using the recombinant strain LP-GFP constructed in step 2), prepare turbot-tracking probiotic feed with fluorescent tracking effect: The prepared LP-GFP was cultured and passaged for more than 20 generations to obtain a stable strain. The stable LP-GFP cells were collected by centrifugation and resuspended in buffer. The bacterial solution was uniformly mixed into commercial turbot feed powder and pelleted feed was prepared. The pelleted feed was aseptically and slowly air-dried to obtain turbot tracer-probiotic feed. 4) Perform initial feeding: Initial feeding phase: Turbot should be fed the above-mentioned turbot tracer-probiotic feed at a daily feeding rate of 1% of their body weight; feeding should be done at 8:00 AM every day; water should be changed 1 / 5 of the way after feeding, and uneaten feed and feces should be removed from the bottom of the pond. 5) Periodic sampling and testing: After 7 days of continuous feeding, samples of experimental turbot were randomly selected for testing; the testing included three dimensions: ① Colonization count detection: The foregut, midgut and hindgut tissues of turbot were separated, the contents of the intestinal lumen were removed, the intestinal lumen was washed with sterile PBS to remove free bacteria, the tissues were ground, and the number of LP-GFP live bacteria adhering to the intestinal tissue was obtained by counting on erythromycin-resistant MRS plates. ② Colonization spatial localization detection: Frozen sections of intestinal tissue were prepared and observed under a fluorescence microscope to determine whether LP-GFP was effectively colonized at the mucus layer-intestinal epithelial interface; ③Intestinal physiological and immune detection: Hematoxylin and eosin (HE) staining of paraffin sections was used to observe the morphology of intestinal villi and mucosa; qRT-PCR was used to detect the expression levels of intestinal IL-1β, TNF-α, IL-10, and occludin genes. 6) Analyze the temporal and spatial colonization patterns of LP-GFP in the foregut-midgut-hingut of turbot to obtain the most important localized target sites; 7) Dynamically adjust the feeding strategy based on the test results. Set a live bacteria adhesion threshold. When the live bacteria adhesion in the intestinal tissue is greater than or equal to the live bacteria adhesion threshold, and LP-GFP is observed to be distributed at the mucus-epithelial interface in fluorescent sections, it indicates that effective colonization has been achieved. Stop feeding turbot tracers-probiotic feed and switch to ordinary commercial feed. Rely on the LP-GFP that has been colonized in the intestine to continue to exert its probiotic effect. Subsequently, samples can be taken at fixed intervals to monitor the colonization decline. When the colonization amount falls below the threshold, feeding can be restarted. When the number of live bacteria adhering to the gut is less than the live bacteria adhesion threshold, the fluorescence signal is weak and effective colonization has not been achieved: continue to feed the turbot tracer-probiotic feed, and repeat the sampling and testing every 3-5 days until the effective colonization threshold is reached.

2. The method as described in claim 1, characterized in that: Steps 1 and 2 are described in detail below: 1) Using pMG36e as the backbone, the GFP gene was fused with the LPXTG anchoring motif derived from Lactobacillus plantarum lp_2578 to construct the recombinant shuttle vector pGLP; 2) pGLP was electroporated into wild-type turbot-derived Lactobacillus plantarum LP-PO23, and recombinant strain LP-GFP was obtained by screening. The strains were continuously passaged in vitro to screen for engineered bacteria with stable plasmids; only strains that passed the passage stability verification were used for turbot tracer-feed preparation.

3. The method as described in claim 1, characterized in that: In step 3), the final concentration of LP-GFP is adjusted to 1×10⁻⁶ by adjusting the bacterial culture concentration. 9 CFU / g was used to obtain turbot tracer-probiotic feed; after preparation, it was stored at -20℃ in a sealed container away from light; before each use, the actual number of viable bacteria was checked by plate counting; if the loss of viable bacteria was >80%, the feed was prepared again.

4. The method as described in claim 1, characterized in that: Step 6) Obtaining the most important fixed target site is as follows: the temporal-spatial colonization pattern is obtained by quantitative counting at multiple time points and localization by fluorescent section.

5. The method as described in claim 4, characterized in that: The multiple time points mentioned in step 6) are 1, 3, 7, 14, and 21 days after feeding was stopped.

6. The method as described in claim 4, characterized in that: The temporal-spatial colonization pattern is as follows: colonization abundance: midgut > foregut > hindgut; with the midgut being the most important colonization target site for LP-GFP.

7. The method as described in claim 6, characterized in that: In step 7), the number of live bacteria adhering to the midgut tissue is ≥10. 4 CFU / g tissue represents the adhesion threshold of live bacteria.

8. The application of the method according to claim 1, characterized in that: The method is applied to the precise feeding of probiotics in the industrialized farming of turbot.

9. The application of the method according to claim 1, characterized in that: The method is used to assess the feeding time required to reach the effective colonization threshold under different aquaculture environments.

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