Lactococcus lactis for promoting intestinal colonization as well as construction method and application thereof

Abstract

The invention discloses lactococcus lactis for promoting intestinal colonization. The lactococcus lactis is recombinant lactococcus lactis capable of expressing flagellin. Accordingly, a corresponding construction method is also established, that is, the flagellin gene is subcloned to a lactococcus lactis expression vector, and then recombinant plasmids are transformed into lactococcus lactis to obtain the lactococcus lactis. Research shows that the recombinant lactococcus lactis with the mFliC3 and the rFliC3 shows an adhesion promoting function in a cell co-culture experiment, and in a sterile mouse experiment, although the bacteria discharge time of the mFliC3 and the rFliC3 is longer than that of an empty vector group, the bacteria discharge time of the rFliC3 is longer than that of the mFliC3. Therefore, both the mFliC3 and the rFliC3 have the function of promoting field planting, and the field planting time of the rFliC3 is longer. Therefore, the intestinal colonization promoting lactococcus lactis disclosed by the invention can be used as probiotics or functional bacteria, is particularly applied to related food or health care products, and can be used for reducing the supplementing times or prolonging the supplementing interval time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of Lactococcus lactis, and particularly relates to a Lactococcus lactis for promoting intestinal colonization, a construction method and application thereof. BACKGROUND

[0002] Lactococcus lactis cells are spherical or oval, gram-positive, facultative anaerobic, do not produce capsules and spores, and have complex nutritional requirements, and the optimum growth temperature is 30 DEG C. Lactococcus lactis is one of the most important and typical strains of lactic acid bacteria, is non-pathogenic to humans and animals, is a potential probiotic, and is widely present in dairy products and plant products, is widely used in the food industry, and is a recognized safe food-grade microorganism.

[0003] At present, most probiotics or functional bacteria cannot colonize in the intestinal tract, so they need to be supplemented frequently, and if they can colonize, the supplementing frequency can be reduced or the supplementing interval can be prolonged. SUMMARY

[0004] The present application aims to solve the technical problem of providing a Lactococcus lactis for promoting intestinal colonization, a construction method and application thereof.

[0005] To solve the above technical problem, the present application adopts the following technical solution:

[0006] The Lactococcus lactis for promoting intestinal colonization is a recombinant Lactococcus lactis capable of expressing flagellin.

[0007] The flagellin is from a flagellin gene.

[0008] The flagellin gene is a mouse SFB flagellin gene M5I-FliC3-2 (mFliC3), a rat SFB flagellin gene R4I-FliC3-1 (rFliC3), or a Salmonella flagellin gene Sal FliC (salFliC3).

[0009] The mouse SFB flagellin gene, the rat SFB flagellin gene, or the Salmonella flagellin gene has the base sequence of the sequence table SEQ.ID.NO. 1 to 3, respectively.

[0010] The construction method of the above Lactococcus lactis for promoting intestinal colonization subclones the flagellin gene into a Lactococcus lactis expression vector, and then transforms the recombinant plasmid into Lactococcus lactis.

[0011] In the construction method, the flagellin gene is a mouse SFB flagellin gene M5I-FliC3-2 (mFliC3), a rat SFB flagellin gene R4I-FliC3-1 (rFliC3), or a Salmonella flagellin gene Sal FliC (salFliC3).

[0012] In the construction method, the mouse SFB flagellin gene, the rat SFB flagellin gene or the Salmonella flagellin gene has the base sequence of the sequence table SEQ.ID.NO. 1 to 3, respectively.

[0013] In the construction method, the Lactococcus expression vector is PAMJ399-eGFP, the Lactococcus is Lactococcus MG1363, and the recombinant plasmid transformation adopts the electric shock transformation mode.

[0014] The above-mentioned Lactococcus promoting intestinal colonization is used as probiotics or functional bacteria.

[0015] Food or health products containing the above-mentioned Lactococcus promoting intestinal colonization.

[0016] In view of the problem that most probiotics or functional bacteria cannot colonize in the intestine, the inventors construct a Lactococcus promoting intestinal colonization, which is a recombinant Lactococcus capable of expressing flagellin. Accordingly, a corresponding construction method is established, that is, the flagellin gene is subcloned into a Lactococcus expression vector, and then the recombinant plasmid is transformed into Lactococcus to obtain it. Studies have shown that the recombinant Lactococcus with mFliC3 and rFliC3 both exhibit adhesion-promoting function in cell co-culture experiments. In the germ-free mouse experiment, although mFliC3 and rFliC3 both have longer bacteria discharge time than the empty vector group, rFliC3 has longer bacteria discharge time than mFliC3. Therefore, mFliC3 and rFliC3 both have a colonization-promoting effect, and the latter has a longer colonization time. Accordingly, the Lactococcus promoting intestinal colonization of the present application can be used as probiotics or functional bacteria, and is particularly applied in related food or health products, which can reduce the supplementing frequency or prolong the supplementing interval. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is a flow chart of a germ-free mouse animal experiment.

[0018] Figure 2 is a monitoring result graph of the growth speed of the recombinant Lactococcus.

[0019] Figure 3 is a result analysis graph of the adhesion of the recombinant Lactococcus and IECs cells, wherein (a)-(d) are samples after the recombinant bacteria are co-cultured with DEME medium for 2 hours, and the sample concentrations are original liquid, 10 -1 , 10 -2 , 10 -3 ; (e)-(h) are samples after the recombinant bacteria and IEC-18 interact for 2 hours, and the sample concentrations are original liquid, 10 -1 , 10 -2 , 10 -3; e, m, r represent Lac-eGFP, Lac-mfliC3 and Lac-rfliC3 respectively.

[0020] Figure 4 is the analysis result diagram of the adhesion of recombinant Lactococcus lactis to MODE-K cells, in which: A and B are the samples after the recombinant Lactococcus lactis is co-cultured with MODE-K cells for 1 hour and 2 hours respectively, and the sample concentrations are the original solution and 10 -1 eGFP, mfliC3 and rfliC3 represent Lac-eGFP, Lac-mfliC3 and Lac-rfliC3 respectively.

[0021] Figure 5 is the electron microscope observation result diagram of the adhesion between Lactococcus lactis and IEC cells, in which: A, B and C represent the Lac-eGFP group, the Lac-mfliC3 group and the Lac-rfliC3 group respectively.

[0022] Figure 6 is the detection result diagram of the excretion of recombinant Lactococcus lactis in feces, in which: A, B and C represent the fecal samples collected from D8, D13 and D17 days respectively; Row 1, Row 2, Row 3 and Row 4 represent different dilution gradients of the fecal samples of the Lac-eGFP group, the Lac-mfliC3 group, the Lac-rfliC3 group and the Lac-salfliC group respectively.

[0023] Figure 7 is the co-localization detection result diagram of rFliC3 and Lamp2, in which: (A) is the DAPI staining result; (B) is the hybridization result with rabbit anti-FliC3 primary antibody and FITC goat anti-rabbit IgG secondary antibody; (C) is the hybridization result with rabbit anti-Lamp2 primary antibody and FITC goat anti-rabbit IgG secondary antibody; (D) is the signal overlapping result of A, B and C.

[0024] Figure 8 is the co-localization detection result diagram of mFliC3 and Lamp2, in which: (A) is the DAPI staining result; (B) is the hybridization result with rabbit anti-FliC3 primary antibody and FITC goat anti-rabbit IgG secondary antibody; (C) is the hybridization result with rabbit anti-Lamp2 primary antibody and FITC goat anti-rabbit IgG secondary antibody; (D) is the signal overlapping result of A, B and C. DETAILED DESCRIPTION

[0025] I. Materials and methods

[0026] 1. Construction and screening of recombinant Lactococcus lactis with flagellin

[0027] Lactococcus lactis MG1363 and E. coli containing PAMJ399-eGFP plasmid were preserved by the applicant. GM17 medium (M17 medium, 0.5% glucose) and LB medium were used for the culture of Lactococcus lactis and E. coli respectively. Lactococcus lactis was incubated statically in an anaerobic workstation at 30°C, and E. coli was incubated in a shaker at 37°C.

[0028] Mouse SFB flagellin gene M5I-FliC3-2 (mFliC3), rat SFB flagellin gene R4I-FliC3-1 (rFliC3) and Salmonella flagellin gene Sal FliC (salFliC3) which the applicant had previously cloned into PMD18-T vector were subcloned into Lactococcus lactis expression vector PAMJ399-eGFP (Reference: Chen H, Yin Y, Wang Y, Wang X, Xiang C. 2017. Host Specificity of Flagellins from Segmented Filamentous Bacteria Affects Their Patterns of Interaction with Mouse Ileal Mucosal Proteins. Appl Environ Microbiol 83.) respectively.

[0029] Preparation of Lactococcus lactis electro-competent cells: a single colony of MG1363 was inoculated into 5 ml solution I (2xM17 50 mL, 50% glucose 1 mL, 50% sucrose 34 mL, 10% glycine 10 mL, ddH20 5 mL) and incubated at 30°C overnight. The bacteria were inoculated into 15 mL solution I at 3-4% inoculation, and incubated at 30°C until OD450=0.4-0.7. The bacteria were collected by centrifugation at 4°C, 5000 rpm for 10 min. The cells were resuspended in 30 mL ice-cold solution II (50% sucrose 34 mL, 87% glycerol 11.5 mL, ddH20 54.5 mL) and centrifuged at 4°C, 5000 rpm for 10 min. The supernatant was removed and the tube was inverted for 1 min to remove the supernatant. The cells were resuspended in 1 mL ice-cold solution II, and incubated at 30-60 min on ice. The cells were aliquoted into Eppendorf tubes pre-cooled at -20°C, and stored at -70°C. The recombinant plasmids were then transformed into Lactococcus lactis MG1363 using an electroporation apparatus (electroporation parameters: 25 μF, 2 KV, 200 Ω). The recombinant bacteria were selected on M17 plates containing erythromycin (final concentration 50 μg / mL), and the recombinant plasmids were verified by sequencing using vector universal primers (P170-F: 5-CCA TTT TTG GTT GCC ATT GTA AC GCT GCC-3 and P170-R: 5-TAC GTA GAT CTG CTC TTC CTG CTT GAG CAT C-3).

[0030] The recombinant Lactococcus lactis expressing flagellin were named Lac-mFliC3, Lac-rFliC3, and Lac-salFliC3, respectively. The empty vector transformed bacteria were named Lac-eGFP.

[0031] 2. Bacterial-cell co-culture and adhesion detection

[0032] IEC-18 cell culture: IEC-18 cells were maintained by the applicant. The cells were cultured in DMEM medium containing 15% fetal bovine serum and 1% double-antibiotic, and subcultured in 12-well plates at a ratio of 1:3 when the cell density reached 80% by first washing the cells with PBS and then trypsinizing the cells. The medium was replaced every 2-3 days to maintain cell viability.

[0033] Bacterial-cell co-culture: Lactococcus lactis was prepared according to the above bacterial culture method, and the bacteria were inoculated into 12-well plates at a density of OD600=0.1-0.2. The IEC-18 cells were subcultured in 12-well plates at a ratio of 1:3, and the bacterial-cell co-culture was performed by adding the bacteria to the IEC-18 cells. 600When the bacterial concentration reaches approximately 1.0, collect the bacterial pellet and dilute it with cell culture medium according to the inoculum size. When cell confluence is nearly 100%, replace the medium with erythromycin-resistant cell culture medium and add the diluted bacterial solution (controlling OD). 600 The values ​​were all around 0.8, and the OD values ​​of all experimental bacterial solutions were adjusted. 600 With consistent values, after mixing the bacterial solutions, 10 μL of the bacterial solution is equivalent to 10 7 (1000 bacteria) were inoculated into the cells, gently shaken to mix, and then the cells were cultured in a tri-gas incubator (5% CO2, 2% O2, 37°C) for 4 hours. After 4 hours of co-culture, the culture supernatant was removed, the cells were gently washed 3 times with PBS, and then the cells and bacteria were repeatedly pipetted and mixed with 1 mL of sterile water. The bacterial-cell mixture was then serially diluted 10-fold, and the different dilutions were inoculated onto erythromycin-resistant M17 plates.

[0034] 3. Recombinant Lactococcus lactis administered to germ-free mice via gavage

[0035] The sterile mouse gavage experiment was conducted at Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. All experimental animals were housed under sterile conditions by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. All procedures were performed in accordance with animal ethics regulations. Fifteen 5-7 week old male C57BL / 6J mice were randomly divided into 5 groups of 3 mice each after one week of environmental acclimatization in biosafety cages. Fecal samples (3-4 pellets / mouse) were collected the day before gavage and stored at -80°C for Gram staining detection. G1 was the saline gavage group (control group), and G2-G5 were administered four different bacterial strains by gavage: Lac-eGFP, Lac-salFliC3, Lac-mFliC3, and Lac-rFliC3, respectively. The dosage was 2 × 10⁻⁶. 8 CFU / mouse / day, administration volume 200 μL. Day 1 of gavage was defined as day 1, and gavage was administered on days 1, 3, and 5. Fecal samples were collected on days 3 and 8 after cessation of gavage for bacterial counting and detection. On day 12 after cessation of gavage, mice were euthanized with CO2, and blood was collected from the heart for serum separation; cecal contents were collected for bacterial counting and detection; liver and terminal ileum tissue (1-2 cm from the cecum) were collected and placed in 1 mL of RNAlater preservation solution, stored overnight at 4°C, and then placed at -80°C.

[0036] 4. Detection of recombinant lactococci in feces

[0037] Fecal samples from monocolony-inoculated mice collected at different time points were weighed and resuspended by shaking at a ratio of fecal weight (mg) / sterile water volume (μL). The resuspended bacterial suspension was serially diluted 10-fold and inoculated into M17 liquid medium and M17 solid medium containing erythromycin, respectively. The samples were then incubated at 30°C in an anaerobic workstation. After 72 hours of incubation, the bacterial growth in each medium was observed. The concentration of erythromycin was 50 μg / mL.

[0038] 5. Immunofluorescence staining and detection

[0039] After animal dissection, the intestinal contents of the terminal ileum were washed away with pre-cooled PBS, and then fixed in 10% formalin. First, the paraffin sections were dewaxed sequentially through xylene, anhydrous ethanol, a series of alcohols, and finally distilled water. Then, antigen retrieval was performed by microwave heating with sodium citrate or EDTA solution at pH 9.0. Next, non-specific binding sites were blocked with 5% BSA, followed by incubation with primary antibody at 4°C overnight. After washing, fluorescently labeled secondary antibody was added, and the sections were incubated in the dark. After washing again, the nuclei were stained with DAPI, and finally, the sections were mounted with resin.

[0040] Throughout the process, it is important to keep the samples moist and avoid drying them to prevent affecting the experimental results. In addition, avoid light exposure during operation to prevent fluorescence quenching. The antibody for mouse SFB flagellate protein FliC3 is a rabbit polyclonal antibody developed by the applicant; the antibody for the late endosome marker LAMP2 protein is a product purchased from the company (DSHB, ABL-93).

[0041] II. Results

[0042] 1. SFB FliC3 has the function of promoting the adhesion of Lactococcus lactis to small intestinal epithelial cells.

[0043] In this invention, the flagellate protein genes of SFB mFliC3 and rFliC3 were first subcloned into the PAMJ399-eGFP expression vector to obtain recombinant plasmids PAMJ399-eGFP-mFliC3 and PAMJ399-eGFP-rFliC3. Then, PAMJ399-eGFP, PAMJ399-eGFP-mFliC3, and PAMJ399-eGFP-rFliC3 were transformed into *Lactococcus lactis* MG1363 by electroporation. Recombinant *Lactococcus lactis* Lac-eGFP, Lac-mFliC3, and Lac-rFliC3 were obtained through antibiotic plate screening and sequencing verification. Bacterial growth curve results (…) Figure 2 The results showed that, compared with the empty PAMJ399-eGFP vector, the introduction of mFliC3 and rFliC3 did not significantly affect the growth rate of recombinant lactococcus, and can be used for cell adhesion experiments.

[0044] Then, the adhesion of recombinant Lactococcus lactis to small intestinal epithelial cells MODE-K (mouse-derived) and IEC-18 (rat-derived) was studied using co-culture. After interaction of Lac-eGFP, Lac-mfliC3, and Lac-rfliC3 recombinant bacteria with DEME medium or IEC-18 for 2 hours, the bacterial culture or cell surface bacterial eluate was inoculated onto M17 solid plates, and colony growth was observed after overnight incubation. After 1 hour and 2 hours of co-culture, the supernatant was removed, sterile water was added, and the cells were repeatedly lysed using a pipette tip. A certain amount of lysis buffer was then spotted onto M17 plates containing erythromycin for Lactococcus lactis culture. Figure 3 As shown, after culturing recombinant Lactococcus lactis in DEME cell culture medium for 2 hours, no significant differences in growth were observed among the groups. However, when these recombinant Lactococcus lactis were co-cultured with IEC-18 cells for 2 hours, the number of bacteria adsorbed on the cell surface showed significant differences. Figure 3 As shown in (g), only 3 colonies of recombinant Lactococcus lactis Lac-eGFP were observed after the cell lysate was diluted 100-fold, while more colonies were observed in recombinant Lactococcus lactis Lac-mFliC3 and Lac-rFliC3. When the cell lysate was diluted 1000-fold and plated, no colonies were observed in the Lac-eGFP group, but 1-2 colonies were still observed in the Lac-mFliC3 and Lac-rFliC3 groups. Figure 3 (f)). Similar results were obtained when these recombinant lactobacilli were co-cultured with MODE-K. Figure 4 As shown, after co-culturing for 1 hour and 2 hours, and diluting the cell lysate 10-fold, the Lac-mFliC3 and Lac-rFliC3 groups showed abundant colony growth, while the Lac-eGFP group had significantly fewer colonies. Simultaneously, the inventors also observed the adhesion of recombinant Lactococcus lactis to the cell surface using electron microscopy. Figure 5 These results indicate that both SFB mFliC3 and rFliC3 promote the adhesion of recombinant bacteria in MODE-K and IEC-18 cells.

[0045] 2. SFB FliC3 has the function of prolonging fecal bacterial excretion in single-implanted mice.

[0046] To further investigate whether SFB mFliC3 and rFliC3 promote the colonization of recombinant Lactococcus lactis in the intestine, the inventors randomly divided 15 male germ-free C57BL / 6J mice aged 6-8 weeks into 5 groups of 3 mice each. In addition to gavage administration of Lac-eGFP, Lac-mfliC3, and Lac-rfliC3 recombinant Lactococcus lactis, respectively, the inventors also set up a control group containing physiological saline and Lac-salfliC3 recombinant Lactococcus lactis (a recombinant bacterium containing Salmonella flagellin). Each mouse in the recombinant bacteria gavage group was administered 200 μL of a solution containing 2 × 10⁻⁶ SFB mFliC3 and rFliC3 daily. 8 The mice in the CFU bacteria saline group were administered 200 μL of sterile saline daily by gavage. Fecal samples were collected one day prior to gavage for Gram staining to confirm sterility before the gavage experiment began. Day 1 was defined as the first day of gavage. Gavage was performed on days 1, 3, and 5, for a total of three gavages. Fecal samples were then collected on days 8, 13, and 17 for plate culture detection of recombinant lactococcus lactis.

[0047] like Figure 6 As shown, on day 8, the third day after gavage was stopped, no recombinant Lactococcus lactis growth was detected in the Lac-eGFP group, but a large number of recombinant Lactococcus lactis could still be detected in the Lac-mfliC3, Lac-rfliC3, and Lac-salfliC3 groups. On day 13, the eighth day after gavage was stopped, only a small amount of bacterial growth could be detected in the Lac-mfliC3 group, but a large number of recombinant Lactococcus lactis could still be detected in the Lac-rfliC3 and Lac-salfliC3 groups. On day 17, the twelfth day after gavage was stopped, no recombinant bacterial growth was detected in the Lac-mfliC3 group, but a large number of recombinant Lactococcus lactis could still be detected in the Lac-rfliC3 and Lac-salfliC3 groups. These results indicate that both SFB flagellin mfliC3 and rfliC3 promote the colonization of recombinant Lactococcus lactis in the intestine, but rfliC3 has a better function in prolonging fecal excretion in single-colonized mice.

[0048] Mouse fecal samples were collected on days 8, 13, and 17. After being diluted with PBS, the samples were spotted onto M17 plates at different dilution gradients. Bacterial growth was observed after 72 hours of bacterial culture.

[0049] 3. Colocalization of SFB recombinant lactococcus with the endocytosis marker Lamp2 was observed.

[0050] Given that Ladinsky et al. reported that SFB introduces antigens into cells via MATE endocytosis, the inventors sought to test whether SFB FliC3 could also be endocytosed. Therefore, after the animal experiments, the inventors collected ileal tissue from mice and used SFB FliC3 polyclonal antibody and Lamp2 polyclonal antibody for immunohistochemical detection. The ileal tissue was fixed in 4% paraformaldehyde and sequentially hybridized with rabbit anti-SFB FliC3 primary antibody, Lamp2 antibody, and FITC goat anti-rabbit IgG secondary antibody. DAPI was used for nuclear staining. Cells were observed using an upright fluorescence microscope (Axio Imager Z2).

[0051] The results showed that the recombinant Lactococcus lactis Lac-rfliC3 could colocalize with the endocytosis marker Lamp2. Figure 7 However, no colocalization of Lac-mfliC3 with the endocytosis marker Lamp2 was detected. Figure 8 ).

Claims

1. A type of lactococcus that promotes intestinal colonization, characterized in that, This bacterium is a recombinant lactococcus that can express flagellin.

2. The *Lactococcus lactis* that promotes intestinal colonization according to claim 1, characterized in that: The flagellin is derived from the flagellin gene.

3. The *Lactococcus lactis* that promotes intestinal colonization according to claim 2, characterized in that: The flagellin gene is the mouse SFB flagellin gene, the rat SFB flagellin gene, or the Salmonella flagellin gene.

4. The intestinal colonization-promoting Lactococcus lactis according to claim 3, characterized in that: The mouse SFB flagellin gene, rat SFB flagellin gene, or Salmonella flagellin gene have the base sequences listed in SEQ ID NO. 1 to 3, respectively.

5. The method for constructing the intestinal colonization-promoting Lactococcus lactis according to claim 1, characterized in that: The flagellin gene was subcloned into a Lactococcus lactis expression vector, and then the recombinant plasmid was transformed into Lactococcus lactis.

6. The construction method according to claim 5, characterized in that: The flagellin gene is the mouse SFB flagellin gene, the rat SFB flagellin gene, or the Salmonella flagellin gene.

7. The construction method according to claim 6, characterized in that: The mouse SFB flagellin gene, rat SFB flagellin gene, or Salmonella flagellin gene have the base sequences listed in SEQ ID NO. 1 to 3, respectively.

8. The construction method according to claim 7, characterized in that: The expression vector for Lactococcus lactis was PAMJ399-eGFP, and the Lactococcus lactis strain was Lactococcus lactis MG1363. The recombinant plasmid was transformed by electroporation.

9. The lactococcus lactis of claim 1, which promotes intestinal colonization, can be used as a probiotic or functional bacterium.

10. Food or health product containing the lactococcus lactis strain described in claim 1 that promotes intestinal colonization.

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