Application of extracellular beta-glucan derived from aureobasidium melanogenum in preparation of training immunity inducer

Abstract

The invention relates to an application of extracellular beta-glucan derived from aureobasidium melanogenum in preparation of a training immunity inducer. By establishing a mouse bone marrow derived macrophages (BMDMs) and zebra fish juvenile fish training immune activation evaluation model, the immunocompetence of extracellular beta-glucan (eBG) derived from aureobasidium melanogenum is evaluated, and it is found that the extracellular beta-glucan has a more remarkable training immune activation effect compared with beta-glucan (cBG) derived from cell walls, and the immunocompetence of the extracellular beta-glucan (eBG) derived from aureobasidium melanogenum is improved. It is proved that the extracellular beta-glucan (eBG) from the melanin-producing aureobasidium is an excellent training immunity inducer and has a good application prospect in the fields of feed addition of bred animals and disease immune prevention and control.

Description

Technical Field

[0001] The present invention relates to the technical field of immune prevention and control, and in particular to the use of extracellular beta-glucan derived from Aureobasidium pullulans in the preparation of a trained immune inducer. Background Art

[0002] Trained immunity is a non-specific immune memory phenomenon. Its basic principle is that the epigenetic modifications produced by immune cells after the initial stimulation are still retained after resting. Therefore, when the cells are stimulated by the same or different pathogens again, a faster and stronger immune response will occur to eliminate the pathogens. Based on the above principle, the activation of trained immunity can enable the host to have a relatively long-term ability to resist pathogenic microorganisms such as bacteria, fungi, viruses or parasites. At present, there are anti-infection or disease immune prevention and control strategies based on trained immunity, for example, using trained immune inducers as immunotherapy drugs, vaccine adjuvants, feed additives, immunopotentiators, etc. As an immunotherapy drug, the activation of trained immunity can enhance the anti-tumor effect, and inhibitors based on the principle of trained immunity can also relieve systemic inflammation. As a vaccine adjuvant, the non-specific immune protection produced by trained immune activation can assist the antigen-specific immune protection effect produced by the vaccine. As a feed additive or immunopotentiator, trained immune inducers can provide long-term protection. For the breeding industry, food-borne supplementation is not only convenient, but also an economical and applicable means of immune protection.

[0003] β-Glucan is a classic training immune inducer, and common sources include bacterial or fungal cell walls, oat or wheat germ, algae cell walls, etc. According to the different ways of linking the main chain, β-glucan is mainly divided into β-1,3-glucan and β-1,4-glucan. On this basis, it may also have side chains mainly connected by β-1,6 glycosidic bonds. However, due to different species and purification methods, cell wall-derived β-glucans have problems such as low purity and poor stability of effects between batches, and the specific branch structure and mechanism of action are also unclear. Previous studies have found that the melanin-producing Acidis fasciculata strain BZW-Δags2-1 / 2, which was modified by genetic engineering, can stably produce extracellular β-glucan (eBG), greatly reducing the cost of β-glucan extraction and purification. eBG belongs to β-(1,3 / 1,6)-glucan with a purity of up to 84.78%. However, there are currently no reports on the evaluation of the effect of eBG on activating training immunity. Summary of the invention

[0004] In order to solve the above problems in the prior art, the present invention provides an application of extracellular β-glucan derived from Aureobasidium pullulans in the preparation of a trained immune inducer.

[0005] In order to achieve the above object, the present invention provides an evaluation of the effect of extracellular β-glucan derived from Aureobasidium pullulans as a trained immune inducer.

[0006] Preferably, the trained immune inducer is used as an immunotherapy drug, a vaccine adjuvant, a feed additive or an immunopotentiator.

[0007] The animal cell training immune activation evaluation model of the present invention is a hematopoietic development process model, which selects β-glucan treatment during macrophage differentiation to promote the maturation of more macrophages. The present invention confirms that the training immune effect induced by eBG is better than that of cell wall β-glucan (cBG) from the same source and β-glucan (BG) from conventional yeast cell walls through the expression level of the pro-inflammatory factor IL-1β in this cell model.

[0008] The in vivo animal training immune activation evaluation model of the present invention is based on the model organism zebrafish. In order to conform to the normal hematopoietic development process of zebrafish, the directional hematopoietic stage with a more lasting effect is selected for the initial stimulation of training immunity, and the zebrafish adaptive immune system has not yet matured during the entire model time.

[0009] As a preferred technical solution of the present invention, the training method on the living model is to use yolk sac injection to simulate eating.

[0010] As a preferred technical solution of the present invention, the infection method of the living model is to use Edwardsiella piscicida injection into the cochlea, and the injection concentration is 50 CFU per fish.

[0011] The present invention confirmed in this in vivo model that eBG enhanced the host's trained immune activation function and produced a substantial anti-infection effect through three aspects: the expression level of pro-inflammatory factors, the evaluation of anti-infection function and the number of macrophages recruited.

[0012] The present invention provides a method for preparing a cell wall-derived β-glucan solution and an extracellular β-glucan solution. The specific implementation method is shown in S2 in Example 1.

[0013] As a preferred technical solution of the present invention, the concentration of cBG and eBG used is 10 μg / mL per well in the cell model and 10 ng per fish in the zebrafish larvae model.

[0014] Conventional trained immune inducers use cell wall-derived β-glucan, and the present invention confirms that extracellular β-glucan has a more obvious trained immune induction effect. The examples provided by the present invention are of great significance for the possibility of extracellular β-glucan replacing cell wall β-glucan for clinical application: on the one hand, extracellular β-glucan can be used as a vaccine adjuvant to enhance the innate immune function of mammals or aquatic animals to achieve the effect of assisting adaptive immunity in long-term protection; on the other hand, extracellular β-glucan can be used as a feed additive to provide a broad-spectrum and long-term anti-infection effect for reducing diseases that occur during the breeding process of mammals or aquatic animals.

[0015] The beneficial effect of the present invention is that by establishing a mouse bone marrow derived macrophage (BMDMs) and zebrafish larvae training immune activation evaluation model, the immune activity of extracellular β-glucan (eBG) derived from Acropora melanocephala is evaluated, and it is found that compared with cell wall-derived β-glucan (BG or cBG), it has a more significant training immune activation effect, which proves that the extracellular β-glucan (eBG) derived from Acropora melanocephala is an excellent training immune inducer and has good application prospects in the fields of feed additives and disease immunity prevention and control in farmed animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of Example 1.

[0017] Figure 2 This is a comparison chart of the IL-1β expression levels induced by eBG, BG and cBG in mouse BMDMs in response to LPS.

[0018] Figure 3 A to Figure 3 C is a comparison of the expression levels of pro-inflammatory factors in mouse BMDMs induced by eBG and cBG in response to Pam3CSK4 and Poly(I:C), respectively.

[0019] Figure 4 A to Figure 4 D is a comparison of the expression levels of pro-inflammatory factors in zebrafish larvae in response to bacterial infection after eBG and cBG treatment, where Figure 4 A is a schematic diagram of Example 3, Figure 4 B to Figure 4 D is the result diagram comparing cBG and eBG.

[0020] Figure 5 This is a comparison of the survival rates of zebrafish larvae under bacterial infection after eBG and cBG treatment.

[0021] Figure 6Comparative evaluation of the bacterial clearance ability of zebrafish larvae after eBG and cBG treatment.

[0022] Figure 7 A and Figure 7 B shows the recruitment of macrophages in the infected cochlea of ​​zebrafish larvae after eBG and cBG treatment. Figure 7 A is the fluorescence result of the zebrafish cochlea, in which green fluorescence marks macrophages. The scale bar is 200 μm. Figure 7 B is a quantitative statistical chart. DETAILED DESCRIPTION

[0023] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.

[0024] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels.

[0025] The present invention provides an application of extracellular β-glucan derived from A. pullulans as a trained immune inducer, which is based on a mouse bone marrow-derived macrophage trained immune model and a zebrafish larvae trained immune activation evaluation model, and proves that the effect of inducing trained immunity of extracellular β-glucan (eBG) derived from A. pullulans is significantly better than that of cell wall β-glucan (cBG) from the same source and conventional yeast cell wall β-glucan (BG) from the same source in terms of pro-inflammatory factor expression level, survival rate, bacterial clearance ability and immune cell recruitment ability. The present invention proposes for the first time that extracellular β-glucan derived from A. pullulans can be used as a trained immune inducer to enhance the host's anti-infection immune response, which is of great significance for guiding the development and application of immune enhancers based on trained immunity.

[0026] The present invention provides an animal cell training immune activation evaluation model, and the model establishment method is shown in Example 1. The present invention provides an animal living body training immune activation evaluation model, and the model establishment method is shown in Example 2.

[0027] The Edwardsiella piscicida (EIB202, CCTCC NO: M208068) used in the present invention is deposited in the Ahua Bioengineering Institute of East China University of Science and Technology.

[0028] The eBG and cBG used in the present invention are both derived from the melanin-producing engineered bacteria BZW-Δags2-1 / 2 (see patent CN117660204A). Conventional yeast-derived BG is purchased from commercial sources.

[0029] Example 1

[0030] eBG enhances mouse BMDM cell training immune activation

[0031] Establishing a mouse BMDMs training immune activation evaluation model includes the following steps:

[0032] S1. Use DMEM medium containing penicillin (100 U / mL) and streptomycin (100 mg / mL) and 10% fetal bovine serum (FBS) at 37°C and 5% CO 2 L929 cells were cultured in a cell culture incubator. After 6-7 days, the cell culture supernatant was collected and filtered with a 0.45 μm filter membrane and stored at 4° C. for later use.

[0033] S2. Dissolve 5 mg of β-glucan in 1 mL of sterile PBS. Ultrasonication conditions: 100 W, 2 s for 2 s and 4 s for 4 s intervals, total ultrasonication time: 1 h. Store at 4 °C for later use.

[0034] S3. Take the hind limbs of 4-6 week old C57 BL / 6 mice, carefully remove the muscles and fascia, keep the intact femur and tibia, cut off the joints, use a 1mL syringe to absorb BMDM culture medium (DMEM+10% FBS+1% double antibody+20% L929 supernatant), preheat at 37°C, insert the needle into the bone marrow cavity to flush the bone marrow into a 50mL centrifuge tube, repeat 3 times until no obvious red color is visible in the leg bone. Repeatedly blow the cells to disperse them, centrifuge at 1000rpm for 10min at 4°C, discard the supernatant and add red blood cell lysis solution (mixed with culture medium in equal proportion), let it stand for 3min, centrifuge under the same conditions and discard the supernatant, reselect the culture medium and sieve it through a 40μm cell filter, and filter it at 3×10 5 Cells were seeded into 24-well plates at a density of 10 cells / well and incubated at 37°C in an atmosphere of 5% CO 2 The cells cultured in a cell culture incubator and adherent to the wall are BMDMs.

[0035] S4. On the third day of culture, the culture medium was replaced with opti-MEM containing 10 μg / mL extracellular β-glucan from Aglaonema pullulans and continued to be cultured as the eBG group, and opti-MEM containing 10 μg / mL cell wall β-glucan from Aglaonema pullulans was used as the cBG group and continued to be cultured. Opti-MEM without additives was used as the control group. After 24 hours, the cells were washed with PBS and replaced with fresh BMDM culture medium for continued culture for 5 days. The culture medium was then replaced with opti-MEM containing 10 μg / mL LPS or 10 ng / mL Pam3CSK4 or 10 ng / mL Poly(I:C) to simulate bacterial infection. After 24 hours, samples were taken for ELISA or qPCR detection.

[0036] S5. Take the cell culture supernatant and perform ELISA to detect the expression level of IL-1β according to the instructions of the kit.

[0037] S6. Take cells to extract RNA, reverse transcribe and perform qPCR detection. The forward primer of the internal reference gene β-actin is: 5'-CATTGCTGACAGGATGCAGAAGG-3', and the reverse primer is: 5'-TGCTGGAAGGTGGACAGTGAGG-3'. The forward primer of the proinflammatory factor TNFA is: 5'-GGTGCCTATGTCTCAGCCTCTT-3', and the reverse primer is: 5'-GCCATAGAACTGATGAGAGGGAG-3'. The forward primer of il6 is: 5'-TACCACTTCACAAGTCGGAGGC-3', and the reverse primer is: 5'-CTGCAAGTGCATCATCGTTGTTC-3'. The forward primer of il1b is: 5'-TGGACCTTCCAGGATGAGGACA-3', and the reverse primer is: 5'-GTTCATCTCGGAGCCTGTAGTG-3'.

[0038] from Figure 2 From the results, we can see that eBG can induce more IL-1β secretion after LPS treatment, and there is no significant difference between BG and cBG, indicating that BG and cBG have similar effects in inducing trained immune activation. Therefore, only cBG and eBG are used for comparison in subsequent experiments. Figure 3 A to Figure 3 The results in C show that the expression level of pro-inflammatory factors in the eBG group increased significantly after secondary stimulation. Compared with the cBG group, eBG treatment not only significantly enhanced the inflammatory response after secondary stimulation of Pam3CSK4, but also significantly enhanced the immune response after secondary stimulation of Poly(I:C).

[0039] Example 2

[0040] eBG enhances the immune activation effect of zebrafish larvae training

[0041] Establishing a zebrafish larvae training immune activation evaluation model includes the following steps:

[0042] S1. Place wild-type AB strain zebrafish into the hatching tank the night before, separate the male and female fish, and remove the partition after the lighting period begins to allow the male and female zebrafish to mate freely. Collect zebrafish embryos and remove impurities by suction. Disinfect with 0.003% sodium hypochlorite for 5 minutes, wash with breeding water three times, each time for 5 minutes, and culture in a constant temperature incubator at 28°C. Remove dead eggs and change water every day.

[0043] S2. When the zebrafish embryos developed to 2 days after fertilization, 2 nl of 5 mg / mL cBG or eBG was injected into the zebrafish yolk sac using a microinjector. Sterile PBS was used as a control group. The injected zebrafish fry were placed in clean system water for static culture.

[0044] S3. After resting for 4 days, when the zebrafish developed to 6 days after fertilization, the fry were anesthetized by sucking into 200 μg / mL tricaine solution, and 50 CFU of Edwardsiella piscicida (E. piscicida) were injected into the cochlea of ​​the zebrafish using a microinjector. Zebrafish samples were taken to extract RNA 3, 8, and 24 hours after injection and infection. 8-10 fry were taken from each tube, and qPCR was performed after reverse transcription. The forward primer of the internal reference gene β-actin was: 5'-GAGCGTAAATACTCCGTCTGG-3', and the reverse primer was: 5'-GACTCATCGTACTCCTGCTTG-3'. The forward primer of the proinflammatory factor TNFA was: 5'-TCAACAAGATGGAAGTGTGCTG-3', and the reverse primer was: 5'-GTCCTGGTCATCTCTCCAGTC-3'. The forward primer of il6 is: 5'-GGCATTTGAAGGGGTCAGGA-3', and the reverse primer is: 5'-GCGTTAGACATCTTTCCGTGC-3'. The forward primer of il1b is: 5'-ATCAAACCCCAATCCACAGAGT-3', and the reverse primer is: 5'-GGCACTGAAGACACCACGTT-3'.

[0045] Figure 4 A to Figure 4 The results of D showed that compared with cBG, eBG could more significantly enhance the expression of pro-inflammatory factors in zebrafish larvae, indicating that eBG has a better training immune activation effect.

[0046] Example 3

[0047] The evaluation of eBG-induced immune activation training to enhance host anti-infection function includes the following steps:

[0048] S1. As in Example 2, 2 nl of 5 mg / mL cBG or eBG was injected into the yolk sac of zebrafish fry 2 days after fertilization, and PBS was used as a control. After resting for 4 days, 50 CFU of Edwardsiella piscicida was injected into the cochlea of ​​the zebrafish fry.

[0049] S2. After infection, observe and count the survival of young fish every 12 hours, discard dead fish in time and change water every day for 3 consecutive days.

[0050] S3. Take the zebrafish larvae swimming normally at 3, 8, and 24 hours after infection for bacterial colonization. Take 3 zebrafish larvae in each tube, discard the culture water, add 100 μl of sterile PBS to each tube, grind with a grinding rod until there is no obvious tissue block, and then dilute the grinding solution in a gradient manner. Dilute 10 1 , 10 2 , 10 3 , 10 4 Then take 10 μl of the original solution or the diluted solution and drop it on the cholethiazide agar plate. Incubate it upside down in a constant temperature incubator at 28℃ for 24 hours and then count the colonies on the plate.

[0051] S4. Calculate the bacterial colonization count results using the following formula:

[0052]

[0053] Figure 5 The results showed that eBG could induce zebrafish larvae to have a higher survival rate under bacterial infection. Figure 6 The results showed that this higher survival rate was associated with stronger bacterial clearance ability, indicating that eBG-induced trained immune activation can enhance the host's anti-infection function.

[0054] Example 4

[0055] eBG-induced immune activation training enhances the anti-infection ability of macrophages, including the following steps:

[0056] S1. Place Tg(mpeg1.1:EGFP) zebrafish in a hatching tank the night before, separate the male and female fish, and remove the partition after the lighting period begins to allow the male and female zebrafish to mate freely. Collect zebrafish embryos and remove impurities by suction. Disinfect with 0.003% sodium hypochlorite for 5 minutes, wash with breeding water three times, each time for 5 minutes, and culture in a constant temperature incubator at 28°C. Remove dead eggs and change water every day.

[0057] S2. When the zebrafish embryos developed to 2 days after fertilization, 2 nl of 5 mg / mL cBG or eBG was injected into the zebrafish yolk sac using a microinjector. Sterile PBS was used as the control group. The injected zebrafish fry were placed in clean system water for static culture.

[0058] S3. After resting for 4 days, when the zebrafish developed to 6 days after fertilization, the fry were anesthetized by suction into a 200 μg / mL tricaine solution, and 50 CFU of Edwardsiella piscicida were injected into the cochlea of ​​the zebrafish using a microinjector. Zebrafish samples were taken for fluorescence imaging 3, 8, and 24 hours after infection.

[0059] S4. 0.5% agarose was used for fixation during fluorescence imaging. The infected cochlea was photographed under a 20x microscope using the Leica Thunder imaging system, and the green fluorescent cells were counted using Image J.

[0060] Figure 7 A and Figure 7 The results in B show that compared with cBG, eBG treatment can recruit more macrophages to the infected site for bactericidal effects in zebrafish larvae.

[0061] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. Application of extracellular β-glucan derived from Acropora melanocarpa in the preparation of trained immune inducers.

2. The use according to claim 1, characterized in that: The trained immune inducer is used as an immunotherapy drug, a vaccine adjuvant, a feed additive or an immunopotentiator.

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