Cetacean bacterium capsular polysaccharide and its application in preventing aquatic viral diseases

Abstract

The application discloses a brycei capsular polysaccharide and application thereof in preventing aquatic viral diseases. 5 Da; the preparation method comprises the following steps: S1, adding an extraction liquid into brycei bacteria bodies, adding a precipitant after stirring, continuously stirring, centrifuging to obtain supernatant A; S2, adding a separation liquid into the supernatant A until no precipitate is generated; adding sterile water, centrifuging to obtain supernatant B; S3, adding a dissociation liquid into the supernatant B, centrifuging to obtain supernatant C, adding a storage liquid into the supernatant C, uniformly mixing, pre-freezing at-70 to-80 DEG C for 2 to 24 hours, and freeze-drying. The brycei capsular polysaccharide can effectively prevent aquatic animal viral diseases, such as viral diseases caused by spring viremia of carp virus, and the effectiveness of the effect is verified through sterile zebra fish and cell models, so that a new thought and means are provided for preventing aquatic viral diseases.

Description

Technical Field

[0001] This invention relates to a capsular polysaccharide of Cetacea and its application in the prevention of aquatic viral diseases, belonging to the fields of life sciences and biotechnology. Background Technology

[0002] Spring viremia of carp virus (SVCV) belongs to the family Rhabdoviridae and the genus Vesicularvirus. It is a single-stranded RNA virus that specifically infects carp, affecting carp of all ages. The mortality rate in infected juvenile carp is extremely high, generally exceeding 70%. SVCV outbreaks cause significant economic losses; therefore, the "List of Class I, II, and III Animal Diseases" includes SVCV in the Class I animal disease list. Simultaneously, due to the widespread prevalence of SVCV in Europe and its serious impact on carp farming worldwide, the World Organisation for Animal Health (OIE) has also listed SVCV as a pathogen requiring mandatory reporting for detection. Currently, there are no effective drugs to treat SVCV. Adding feed additives to enhance fish immunity is one effective way to combat SVCV. Probiotics are live microorganisms that, at appropriate doses, have beneficial effects on the host's health. Numerous studies have confirmed the use of feed microorganisms to improve fish growth performance and enhance their immune resistance; cetacean is a probiotic strain for aquatic animals. Metabiotics are preparations of non-living microorganisms and / or their components that are beneficial to the host's health. Capsular polysaccharides are a major component of the cell wall capsule and one of the leading representatives of the new generation of metabiotics. Studies have found that, compared to live single strains, capsular polysaccharides from certain metabiotic strains can better restore gut microbiota diversity, avoiding the safety risks associated with introducing single strains and exhibiting better safety and efficacy. Furthermore, compared to live strains, metabiotics with specific activities are easier to preserve and promote. Therefore, the development of novel strain metabiotics has significant market value. Summary of the Invention

[0003] The purpose of this invention is to provide a cetacean and its application in the prevention and treatment of aquatic viral diseases. This invention is the first to discover the preventive effect of cetacean capsular polysaccharide, especially the preventive effect of cetacean capsular polysaccharide on aquatic viral diseases, and the effectiveness of this effect has been verified by sterile zebrafish and cell models, thus providing a new idea and means for the prevention of aquatic viral diseases.

[0004] The cetacean capsular polysaccharide provided by this invention has a weight-to-number-average molecular weight ratio of 1.04 and a number-average molecular weight of 4.12 × 10⁻⁶. 5 Da.

[0005] The cetacean capsular polysaccharide provided by this invention is composed of glucuronic acid, rhamnose, glucose, glucosamine hydrochloride and galactose, with a molar ratio of 58.7:16:11.9:9.3:4.1.

[0006] The present invention also provides a method for preparing the cetacean capsular polysaccharide, comprising the following steps:

[0007] S1. Add the extract to the cetobacter cells, stir, add the precipitant, continue stirring, and centrifuge to obtain supernatant A;

[0008] S2. Add separation liquid to the supernatant A until no precipitate is found; add sterile water and centrifuge to obtain supernatant B;

[0009] S3. Add dissociation solution to the supernatant B, centrifuge to obtain supernatant C, add preservation solution to supernatant C, mix well, pre-freeze at -70 to -80°C for 2 to 24 hours, and then freeze-dry.

[0010] In the above preparation method, in step S1, the extract can be a GENMED extract;

[0011] The precipitant may be a gentmed precipitant;

[0012] The centrifugation conditions are: speed of 1000-3000g and time of 60-120min.

[0013] In the above preparation method, step S1 further includes the following step of treating the precipitate obtained by centrifugation:

[0014] Add the extract to the precipitate, vortex and centrifuge, and mix the resulting supernatant with supernatant A;

[0015] The centrifugation conditions are: speed of 1000-3000g, time of 60-120min;

[0016] The separation solution is a GENMED separation solution.

[0017] In the above preparation method, if no precipitate is formed in step S2, then

[0018] In the above preparation method, in step S3, the dissociation solution is a GENMED dissociation solution;

[0019] The centrifugation conditions are: speed of 1000-3000g, time of 30-60min;

[0020] The supernatant C was divided into two portions, and ethanol was added to each portion for incubation. After centrifugation, ethanol was added to the supernatant again for incubation. After centrifugation, the supernatants were combined.

[0021] The preservation solution is a GENMED preservation solution.

[0022] The cetacean capsular polysaccharide provided by this invention can effectively prevent viral diseases in aquatic animals, such as viral diseases caused by carp spring viremia virus. The effectiveness of this effect has been verified through sterile zebrafish and cell models, thus providing a new approach and means for the prevention of viral diseases in aquatic animals. Attached Figure Description

[0023] Figure 1 This is the capsule structure of *Cetacea* in Example 1 of the present invention.

[0024] Figure 2 This is an image of the extraction of CPS capsular polysaccharide from Cetacea bacillus in Example 2 of the present invention.

[0025] Figure 3 The calibration curves for lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight) of cetacean capsular polysaccharide in Example 3 of the present invention are shown.

[0026] Figure 4 This is the molecular weight spectrum of CPS capsular polysaccharide from Cetacea in Example 3 of the present invention.

[0027] Figure 5 This is the ion chromatogram of the mixed standard 16 sugar in Example 3 of the present invention (mixed standard: solvent peak: sodium hydroxide peak at 2.0 min, sodium acetate peak at 40.5 min).

[0028] Figure 6 The monosaccharide composition of CPS capsular polysaccharide in *Cetacea* was determined by ion chromatography in Example 3 of this invention.

[0029] Figure 7 The results of detecting the anti-SVCV virus activity of Cetacea capsular polysaccharide CPS in the ZF4 cell model in Example 4 of this invention are shown.

[0030] Figure 8 The results of detecting the anti-SVCV virus activity of Cetacea capsular polysaccharide CPS in a sterile zebrafish model in Example 5 of this invention are shown. Detailed Implementation

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0034] In the following examples, *Cetobacterium somerae* XMX-1 (CGMCC No. 18908) was cultured on Gifu Anaerobic Medium (GAM) at 28°C in an anaerobic incubator for 12 hours. *Cetobacterium somerae* XMX-1 is deposited at the China General Microbiological Culture Collection Center. In the following text, *Cetobacterium somerae* XMX-1 is abbreviated as *Cetobacterium*. *Cetobacterium* capsular polysaccharide is abbreviated as CPS.

[0035] Tu strain zebrafish (described in the following literature: Loucks E, Carvan III M J. Strain-dependent effects of developmental ethanol exposure in zebrafish. Neurotoxicology and teratology, 2004, 26(6):745-755.) were provided by the Zebrafish Aquatic Laboratory of Peking University. In the following text, Tu strain zebrafish were used for the preparation of sterile zebrafish.

[0036] The methods for producing, culturing, and transferring germ-free (GF) zebrafish are based on those proposed by Pham et al. (described in the following literature: Pham LN, Kanther M, Semova I, et al. Methods for generating and colonizing gnotobiotic zebrafish. Nature protocols, 2008, 3(12): 1862.) and the improvements made by the applicant team (Ran C, Hu J, Liu W, et al. Thymol and carvacrol affect hybrid tilapia through the combination of direct stimulation and an intestinal microbiota-mediated effect: insights from a germ-free zebrafish model. The Journal of nutrition, 2016, 146(5): 1132-1140.).

[0037] The quantitative real-time PCR analysis was performed using the SuperReal PreMix Plus kit (a product of Tiangen Pharmaceuticals).

[0038] Cells used: Zebrafish embryonic fibroblast ZF4 cell line was purchased from the American Type Culture Collection Center (ATCC).

[0039] ZF4 cells (zebrafish embryonic fibroblasts) were purchased from the American Collection of Cell Cultures (ATCC: CRL-2050) and preserved at the China-Norway Joint Laboratory of Fish Digestive Tract Microbiology, Institute of Feed Research, Chinese Academy of Agricultural Sciences (hereinafter referred to as "this laboratory"), and cultured in a 28°C, 5% CO2 incubator. EPC cells (ATCC CRL-2872 carp epithelial cell line) and SVCV (carp spring viremia virus, ATCC: VR-1390) were donated by Professor Yuan Junfa (Huazhong Agricultural University, Wuhan, China). EPC cells were cultured in a 28°C, 5% CO2 incubator.

[0040] SVCV preparation method: SVCV proliferation was performed using EPC cells. The EPC cell culture medium was Minimum Essential Media (MEM) containing 10% fetal bovine serum, and the culture conditions were 25°C and 5% CO2. Specifically, the method involved using cryopreserved SVCV virus seeds (approximately 10...) 8 TCID 50 Dissolve the virus ( / ml) on ice, and take 4 μl into 10 ml of cell culture medium. Then, transfer the virus-containing MEM medium to EPC cells layered in a culture dish and culture until 80% of the cells show pathogenic effects. Transfer the culture dish to -80°C. Repeat the freeze-thaw cycle three times to release the virus from the cells. Finally, centrifuge the cell lysis buffer at 12000g at room temperature for 20 min, collect the supernatant, and transfer it to a sterile tube for storage. (TCID virus) 50 The method for determining (tissue median infectious dose) was performed according to the previously published protocol (Li, Y., Ran, C., Wei, K., Xie, Y., Zhou, Z., The effect of astragalus polysaccharide on growth, gut and liver health, and antiviral immunity of zebrafish. Aquaculture, 2021:736677.).

[0041] Example 1: Capsular structure of Cetacea

[0042] The capsule is a specialized bacterial structure, a liquid substance that surrounds the cell wall of some bacteria. Its components are polysaccharides, glycoproteins, or polypeptides. The chemical composition of the capsule has a weak binding affinity for dyes, making it difficult to stain, and it is water-soluble and easily removed during washing. Therefore, a contrast staining method (negative staining) is generally used to stain the bacterial cells and background, while the capsule remains unstained, forming a clear ring around the bacterial cells (i.e., the capsule). The Tyler method was used to stain the capsule of *Cetacea*.

[0043] Ingredients: 20% CuSO4 aqueous solution (to stain the background blue-purple), crystal violet (to stain the bacterial cells purple);

[0044] Smear preparation: Prepare smears using the conventional method. Take a large amount of bacterial cells and mix them thoroughly with water. Spread the viscous bacterial solution as widely as possible, but do not cover too large an area.

[0045] Drying: air drying;

[0046] Staining: Stain with Tyler's staining solution for 5-7 minutes;

[0047] Decolorization: Wash away crystal violet with 20% CuSO4 aqueous solution. Decolorization should be moderate (rinse twice) → blot dry with absorbent paper → immediately add 1-2 drops of cedar oil to the smear to prevent the formation of CuSO4 crystals;

[0048] Microscopic examination: Observe first under low magnification and then under high magnification.

[0049] Using a cetacean capsule staining method, the background is blue-purple, the cetacean cells are purple, and the cetacean capsule is colorless or light purple, indicating that cetacean has a capsule structure. Figure 1 As shown.

[0050] Example 2: Extraction of capsular polysaccharides from Cetacea

[0051] A capsular polysaccharide purification kit was purchased from Jemei Gene Company to prepare 50L of exponential growth phase (6.3×10⁻⁶). 8Add 500 ml of GENMED extraction buffer (Reagent A) to cetacean bacteria (CFU / ml) and mix well. Transfer to a 1-liter beaker and stir magnetically for 60 min. Add 50 g of GENMED precipitant (Reagent B) and continue stirring magnetically for 60 min until the liquid is homogeneous. Transfer to the appropriate centrifuge tube. Centrifuge at 1000 g for 120 min at room temperature. Carefully transfer the supernatant to a new 1-liter beaker. Add 50 ml of GENMED extraction buffer (Reagent A) to the precipitate tube, vortex for 1 min to mix the particles, and transfer to the appropriate centrifuge tube. Centrifuge at 1000 g for 120 min at room temperature. Carefully transfer the supernatant to the prepared 1-liter beaker (combined) and stir magnetically. Simultaneously, add GENMED separation buffer (Reagent C) drop by drop until no precipitate is observed. Transfer the buffer to the appropriate centrifuge tube and centrifuge for 15 minutes at 1000g. Carefully remove the supernatant. Add 50ml of sterile dd H2O, mix the particle group, centrifuge for 15 minutes at 1000g, and carefully remove the supernatant. Repeat the above steps once. Add 300 ml of GENMED dissociation buffer (Reagent D), mix the particle group, stir magnetically for 30 min, transfer to the corresponding centrifuge tubes, centrifuge for 15 min at 1000 g, carefully transfer the supernatant to two 1-liter beakers (150 ml each), add 450 ml of 95% ethanol to each, mix well, incubate at -20°C for 60 min, centrifuge for 30 min at 1000 g, carefully remove the supernatant, add 50 ml of 95% ethanol to each, centrifuge for 30 min at 1000 g, carefully remove the supernatant, repeat the above steps twice, combine and finally add 22.5 ml of GENMED preservation buffer (Reagent E), mix well, pre-freeze at -80°C, freeze-dry after 12 h.

[0052] The results showed that the amount of capsule polysaccharide precipitate after extraction and purification of 50L of *Cetacea* cells was as follows: Figure 2 As shown in Figure A, the polysaccharide was dissolved in 22.5 ml of GENMED preservation solution, pre-frozen at -80℃, and then freeze-dried after 12 hours. The completely freeze-dried product is shown in Figure A. Figure 2 As shown in Figure B, 20 mg of capsular polysaccharide was obtained, and the yield of capsular polysaccharide was 40 mg / L (yield = sugar harvest mass (mg) / fermentation broth volume (L)).

[0053] Example 3: Determination of molecular weight and monosaccharide composition of cetacean capsular polysaccharide

[0054] The structure of the cetacean capsular polysaccharide extracted by Borui Sugar Biotechnology Co., Ltd. in Example 2 was determined. The molecular weight and purity of the polysaccharide were determined by HPGPC, and the monosaccharide composition was determined by ion chromatography.

[0055] Molecular weight test results as follows Figure 3 and Figure 4 As shown, the calibration curves for lgMp-RT (peak molecular weight), lgMw-RT (weight-average molecular weight), and lgMn-RT (number-average molecular weight) were obtained. The equation for the lgMp-RT calibration curve is: y = -0.238x + 11.86, R 2 =0.99; The equation of the lgMw-RT correction curve is: y = -0.243x + 12.03, R 2 =0.990; the equation of the lgMn-RT correction curve is: y = -0.238x + 11.88R 2 =0.99; Based on the standard curve, the calculation formula was derived to calculate the molecular weight of each sample. The calculation results are shown in Table 1 below.

[0056] Table 1. Molecular weight of the samples

[0057] 26.32 5.60 5.63 5.62 394312 430765 412895 100

[0058] (Note: Mn is the number-average molecular weight, Mp is the peak molecular weight, Mw is the weight-average molecular weight. The unit of molecular weight is Dalton. The peak at 38.3 min is the solvent peak.)

[0059] The results showed that the ratio of weight-average molecular weight to number-average molecular weight (α = Mw / Mn) of the extracted *Cetacea* capsular polysaccharide was 1.04, indicating that the extracted *Cetacea* capsular polysaccharide was a single polysaccharide with a molecular weight of approximately 4.12 × 10⁻⁶. 5 Da.

[0060] Next, 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannulic acid) were prepared into standard stock solutions. Precise concentration standards were prepared from each monosaccharide standard solution to create mixed standards. The mass of different monosaccharides was determined using an absolute quantification method, and the molar ratio was calculated based on the molar mass of the monosaccharides. 5 mg of *Cetacea* capsular polysaccharide was accurately weighed and placed in an ampoule, 2 mL of 3 MTFA was added, and hydrolysis was carried out at 120 °C for 5 h. The acid-hydrolyzed solution was accurately transferred to a tube, dried under nitrogen, and 5 mL of water was added and vortexed. 500 μL of the solution was then added to 500 μL of deionized water and centrifuged at 12000 rpm for 5 min. The supernatant was then analyzed by IC50.

[0061] Table 2 Monosaccharide composition of cetacean capsular polysaccharide

[0062]

[0063] The monosaccharide composition of the extracted cetacean capsular polysaccharide was identified, and it was found that the cetacean capsular polysaccharide was composed of glucuronic acid, rhamnose, glucose, glucosamine hydrochloride and galactose, and its molar ratio was 58.7:16:11.9:9.3:4.1.

[0064] Figure 5 The image shows the ion chromatogram of a mixed standard 16-sugar mixture (mixed standard: solvent peaks: sodium hydroxide peak at 2.0 min, sodium acetate peak at 40.5 min). Figure 6 To determine the monosaccharide composition of CPS capsular polysaccharide from Cetacea using ion chromatography.

[0065] Example 4: Detection of antiviral activity of cetacean capsular polysaccharide in cell model

[0066] With a density of 1×10 6 ZF4 cells were seeded in 6-well cell culture plates and cultured at 28°C. When the cells reached 80% confluence, 0, 0.25 ug / ml, and 2.5 ug / ml of Cetacea capsular polysaccharide CPS extracted in Example 2 were added. After 24 hours, the cells were washed three times with DMEM / F12 medium and then 10 μg / ml of CPS was added. 6 TCID 50 ZF4 cells were incubated with SVCV at 28°C for 1 hour, then the medium was replaced with DMEM / F12 containing 2% fetal bovine serum. After 24 hours of culture, TRIzol reagent was added to extract total RNA from each sample, which was reverse transcribed into cDNA. Quantitative real-time PCR was then used to analyze the expression level of the SVCV N gene in the cDNA of ZF4 cells to indicate viral load. The primers for detecting the internal control RPS11 gene were 5'-ACAGAAATGCCCCTTCACTG-3' and 5'-GCCTCTTCTCAAAACGGTTG-3'.

[0067] The primers used to detect the SVCVN gene are 5'-TGAGGTGAGTGCTGAGGATG-3' and 5'-CCATCAGCAAAGTCCGGTAT-3'.

[0068] The results showed that, compared with the control group, the purified *Cetacea* capsular polysaccharide at 2.5 μg / ml had a significant antiviral effect. Figure 7 As shown.

[0069] Example 5: Detection of antiviral activity of Bacillus cetacea capsular polysaccharide in a sterile zebrafish model

[0070] Live morpholino oligonucleotides (MOs) of Myd88 were designed and synthesized using Gene Tools. Their MO sequences are as follows: CK-MO: 5-CCTCTTACCTCAGTTACAATTTATA-3; Myd88-MO (translating blocking): 5-ATATCCACAAAGCAACATGCCTTTT-3. Four days post-fertilization (4 dpf), when all germ-free (GF) zebrafish had emerged from the membrane, 2.5 μg / ml of the *Cetaceae* capsular polysaccharide CPS extracted in Example 2 was added, along with CK-MO / Myd88-MO, to a final concentration of 50 nM. The zebrafish from each group were then transferred to a 25°C light incubator overnight. After 7 dpf, 90% of the liquid in the vial was aspirated, and 10 mL of GZM was added. For the experimental groups, 10 mL of GZM was added. 6 TCD50 / mL SVCV virus solution was added to the control group, and the same volume of MEM medium was added. At 9 dpf, whole juvenile fish were collected, and 20 juvenile fish were mixed into one sample. RNA was extracted and reverse transcribed to obtain zebrafish cDNA. The expression level of the SVCV N gene in the zebrafish cDNA was then analyzed by real-time quantitative PCR to indicate the viral load.

[0071] The results showed that the GF+CPS group had significant antiviral activity compared to the GF group; however, compared to the CK-MO group, CPS showed no significant antiviral activity after Myd88 MO knockdown. This indicates that CPS exhibits antiviral activity, but this activity is dependent on the Myd88 signaling pathway. Figure 8 As shown.

Claims

1. A capsular polysaccharide of *Cetacea*, having a weight-to-number-average molecular weight ratio of 1.04 and a number-average molecular weight of 4.12 × 10⁻⁶. 5 Da; The cetacean capsular polysaccharide is composed of glucuronic acid, rhamnose, glucose, glucosamine hydrochloride and galactose in a molar ratio of 58.7:16:11.9:9.3:4.

1. The method for preparing the cetacean capsular polysaccharide includes the following steps: S1. Add the extract to the cetobacter cells, stir, add the precipitant, continue stirring, and centrifuge to obtain supernatant A; The precipitant is a GENMED precipitant; The cetobacter is cetobacter. Cetobacterium somerae XMX-1, with accession number CGMCC No.18908; S2. Add separation liquid to the supernatant A until there is no precipitate, centrifuge to remove the supernatant, add sterile water, and centrifuge to obtain the precipitate; The separation solution is a GENMED separation solution; S3. Add dissociation solution to the precipitate obtained in step S2, centrifuge to obtain supernatant C, add ethanol for incubation, centrifuge, remove the supernatant and add ethanol again for incubation, centrifuge, remove the supernatant, then add preservation solution, mix well, pre-freeze at -70~-80°C for 2~24 h and freeze dry. The dissociation solution is a GENMED dissociation solution; The precipitant, separation solution, and dissociation solution were obtained from the capsular polysaccharide purification kit of JMEG Biotech.

2. The method for preparing the *Cetacea* capsular polysaccharide according to claim 1 comprises the following steps: S1. Add the extract to the cetobacter cells, stir, add the precipitant, continue stirring, and centrifuge to obtain supernatant A; The precipitant is a GENMED precipitant; The cetobacter is cetobacter. Cetobacterium somerae XMX-1, with accession number CGMCC No.18908; S2. Add separation liquid to the supernatant A until there is no precipitate, centrifuge to remove the supernatant, add sterile water, and centrifuge to obtain the precipitate; The separation solution is a GENMED separation solution; S3. Add dissociation solution to the precipitate obtained in step S2, centrifuge to obtain supernatant C, add ethanol for incubation, centrifuge, remove the supernatant and add ethanol again for incubation, centrifuge, remove the supernatant, then add preservation solution, mix well, pre-freeze at -70~-80°C for 2~24 h and freeze dry. The dissociation solution is a GENMED dissociation solution; The precipitant, separation solution, and dissociation solution were obtained from the capsular polysaccharide purification kit of JMGene.

3. The preparation method according to claim 2, characterized in that: In step S1, the extract is a GENMED extract; The centrifugation conditions are: speed of 1000~3000 g, time of 60~120 min.

4. The preparation method according to claim 2 or 3, characterized in that: In step S1, the method further includes the step of treating the precipitate obtained by centrifugation as follows: Add the extract to the precipitate, vortex and centrifuge, and mix the resulting supernatant with supernatant A; The centrifugation conditions are: speed of 1000~3000 g, time of 60~120 min.

5. The preparation method according to claim 2 or 3, characterized in that: In step S3, the centrifugation conditions are: speed of 1000~3000 g, time of 30~60 min; The preservation solution is a GENMED preservation solution.

6. The use of the cetacean capsular polysaccharide of claim 1 in the preparation of a medicament for the prevention and / or treatment of viral diseases in aquatic animals; The aquatic animal viral disease is caused by carp spring viremia virus.

7. A drug for the prevention and / or treatment of viral diseases in aquatic animals, wherein the active ingredient is the cetacean capsular polysaccharide as described in claim 1; The aquatic animal viral disease is caused by carp spring viremia virus.

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