Egg yolk antibody-nano chitosan conjugate and application thereof

Abstract

The invention relates to a conjugate formed by an egg yolk antibody and nano chitosan, and a pharmaceutical composition or a feed composition containing the conjugate, the conjugate can improve or eliminate inflammatory response of human or animal bodies, and can regulate intestinal flora so as to play a role in protecting intestinal mechanical barrier and immune barrier functions.

Description

Technical Field

[0001] The invention relates to a conjugate of specific egg yolk antibody and nano chitosan, a pharmaceutical composition containing the conjugate and the medicinal use thereof. Background Art

[0002] Yolk immunoglobulin Y (Immunoglobulin Y, IgY), also known as yolk antibody, has a molecular weight of about 180kDa and is composed of 2 light chains and 2 heavy chains, of which the molecular weight of the light chain is about 22-30kDa and the molecular weight of the heavy chain is about 67-70kDa. IgY is a specific immunoglobulin produced by B lymphocytes and gradually accumulated in the yolk of poultry after antigen stimulation. As an effective passive immune antibody preparation, IgY is considered to be the functional equivalent of mammalian immunoglobulin G (Immunoglobulin G, IgG). It has the advantages of being non-toxic, non-residual, and not causing bacteria to develop drug resistance. It has been widely used in the prevention and treatment of human and animal diseases. Chitosan (CS), also known as chitosan, is a deacetylated derivative of chitin and is the alkaline polysaccharide found in nature so far. It has excellent antibacterial properties, excellent strong adsorption, strong moisture absorption and water retention, and is a natural biomass material with a wide source, low price and easy to obtain. Nanochitosans (CNs) are cross-linked chitosan derivatives that were originally developed as drug carriers.

[0003] Inflammation is a spontaneous reaction of the body to maintain its own homeostasis caused by the invasion of external viruses or chemicals. Many molecules involved in the early stage of immune response and various stages of inflammatory response are regulated by NF-κB, including: TNF-α, IL-1β, IL-2, IL-6, IL-8, IL-12, iNOS, COX2, chemokines, adhesion molecules, colony stimulating factors, etc. Among them, NF-κB, TNF-α, IL-6, and IL-1β are all multi-cell source factors, and play an important role in the occurrence and transformation of many diseases. Nuclear transcription factor NF-κB is a multi-subunit nuclear transcription factor that plays an important role in the regulation of many genes, including participation in immunity and inflammation, promotion or inhibition of chemotaxis and expression of related apoptosis proteins, cell proliferation, and tumorigenesis. Studies have shown that when cells are induced by factors such as inflammatory mediators or oxidative stress, NF-κB enters the cell nucleus to regulate the expression of cytokines and inflammatory mediators, thereby regulating the physiological and pathological processes of tissues and cells.

[0004] Increasing the intake of unsaturated fatty acids, vitamin E, dietary fiber and fresh fruits and vegetables can help control the occurrence of inflammation. In addition to the physiological regulation of these dietary nutrients, the prevention and treatment of inflammation by non-nutritional ingredients has also become a new research hotspot.

[0005] In addition, in animal husbandry, for example, early weaning of piglets can easily lead to weaning stress, induce intestinal flora disorders, intestinal barrier damage and intestinal inflammation, leading to diarrhea in piglets and even death, seriously endangering the healthy breeding of pigs. In recent decades, weaning stress, intestinal inflammation and diarrhea in piglets have been a problem that has troubled pig farmers and an important research topic for animal husbandry scholars. Summary of the invention

[0006] Based on the requirements of the prior art, the object of the present invention is to provide a conjugate of specific egg yolk antibody and nano-chitosan, and its medicinal activity on humans and animals.

[0007] A conjugate is formed by covalently coupling egg yolk antibody and chitosan through amide bonds formed by carboxyl groups and amino groups.

[0008] According to the conjugate of the present invention, the chitosan is nano-chitosan.

[0009] According to the conjugate of the present invention, the egg yolk antibody is a specific egg yolk antibody specific to the drug-resistant Escherichia coli antigen.

[0010] The method for preparing the specific yolk antibody of the present invention is known to those skilled in the art. In one embodiment of the present application, laying hens are immunized with drug-resistant Escherichia coli antigens, and yolk antibodies are extracted from the yolk fluid using polyethylene glycol precipitation.

[0011] According to the conjugate of the present invention, the nano-chitosan and the egg yolk antibody form a covalent coupling under the action of the cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS).

[0012] According to the conjugate of the present invention, the egg yolk antibody and nano-chitosan are mixed in a sodium acetate buffer at a weight ratio of 5-10:1-3 for covalent coupling.

[0013] According to the conjugate of the present invention, the conjugation is reacted at 0-8°C for 10-30 hours.

[0014] The conjugate according to the present invention is freeze-dried to prepare nanoparticles.

[0015] The present invention also provides a pharmaceutical composition comprising the above conjugate, which comprises the above conjugate and pharmaceutically acceptable excipients.

[0016] The present invention also claims a feed additive, which contains the above-mentioned conjugate and acceptable auxiliary materials.

[0017] The above-mentioned pharmaceutical composition or feed additive according to the present invention is used for humans or animals, especially for livestock animals.

[0018] The present invention also provides the use of the conjugate for preparing a drug with anti-inflammatory activity.

[0019] According to the use of the present invention, the anti-inflammatory activity is to inhibit or alleviate the reduction of IPEC-J2 cell proliferation activity caused by inflammatory response.

[0020] According to the use of the present invention, the anti-inflammatory activity is to reduce the inflammatory response of IPEC-J2 cells through the NF-κB signaling pathway.

[0021] According to the use of the present invention, the anti-inflammatory activity is to alleviate the inflammatory damage of IPEC-J2 cells induced by LPS by regulating the secretion of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α.

[0022] The present invention also provides another use of the above-mentioned conjugate, which is used for preparing a medicine or feed additive for regulating the intestinal microbial ecology of humans or animals.

[0023] According to the use of the present invention, regulating the intestinal microbial ecology refers to promoting lactic acid fermentation, improving intestinal mucosal health and / or immune regulation.

[0024] According to the use of the present invention, the intestinal microbial ecology is regulated by increasing the abundance of bacteria including but not limited to the following, thereby improving the health of the intestinal mucosa, improving the intestinal barrier function, and immune regulation: Bacillus (c__Bacilli), Lactobacillaceae (f__Lactobacillaceae), Lactobacillus (g__Lactobacillus), Lactobacillales (o__Lactobacillales), Verrucomicrobiae (c__Verrucomicrobiae), Akkermansia (g__Akkermansia), Verrucomicrobiaceae II (f__Akkermansiaceae), Verrucomicrobiota (p__Verrucomicrobiota), Verrucomicrobiales (o__Verrucomicrobiales), Faecalibaculum (g_Faecalibaculum), etc.

[0025] In the present invention, the animal body, the livestock animal, is, for example, a pig, a sheep, a cow, a chicken, and especially a piglet.

[0026] Beneficial Effects

[0027] In the prior art, chitosan is used as a wall material carrier and an ion crosslinking method is used to prepare microcapsules of yolk antibodies, and then the effect of the medicinal form of the microcapsules compared with the single yolk antibody is studied. However, the prior art has never reported a new substance, a conjugate of yolk antibodies and nano-chitosan, and the synergistic effect of the two on new medicinal or biological values. The present application has developed a new chemical conjugate of yolk antibodies and nano-chitosan, and found that the conjugate can improve or eliminate the inflammatory response of cells and animals, can regulate the intestinal flora of animals, and thus play a role in protecting the intestinal mechanical barrier and immune barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : FTIR spectra of CNs and IgY-CNs conjugates;

[0029] Figure 2 :The effect of IgY-CNs on the relative expression of genes related to TLR4 / NF-κB signaling pathway in IPEC-J2 cells under LPS stimulation;

[0030] Figure 3 :The effect of IgY-CNs on the relative expression of intestinal barrier-related genes mRNA in IPEC-J2 cells under LPS stimulation;

[0031] Figure 4 :The effect of IgY-CNs on the protein expression of NF-κB pathway in IPEC-J2 cells stimulated by LPS;

[0032] Figure 5 : Relative abundance of mouse cecal microorganisms at the phylum level;

[0033] Figure 6 : Relative abundance of mouse cecal microorganisms at the genus level;

[0034] Figure 7 :Differential microorganisms at the cecal level in mice;

[0035] Figure 8 :Differential microorganisms at the genus level in the mouse cecum;

[0036] Fig. 9 : Lefse multilevel species difference discrimination analysis, A. Differential bacteria between the control group and the low-dose group; B. Differential bacteria between the control group and the medium-dose group; C. Differential bacteria between the low-dose group and the medium-dose group. DETAILED DESCRIPTION

[0037] The conjugate and application of the present invention will be described in further detail below in conjunction with specific examples. It should be understood that the following examples are only exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0038] Statistical analysis: In the present invention, the results are expressed as mean ± standard error (SEM). SPSS26.0 software was used to perform one-way analysis of variance on the data, and Duncan's multiple comparisons were used to determine the differences between different treatments and plot them. P < 0.05 was considered statistically significant.

[0039] Example 1 Preparation of yolk antibody-chitosan nanoparticle conjugate

[0040] (1) Preparation of yolk antibody IgY

[0041] Healthy Hy-Line brown laying hens one month before laying were selected. The test subjects were divided into two groups, with 10 laying hens in each group. The test group was injected with 2×10 9 CFU / mL resistant Escherichia coli antigen was used to immunize laying hens, with an immunization dose of 1mL / hen (multiple injections, about 0.2mL per point) to obtain specific IgY. The control group was injected with an equal amount of sterile PBS buffer into the pectoral muscle to obtain nonspecific IgY. Additional immunizations were performed at weeks 3, 5, and 7 after the initial immunization. Freund's complete adjuvant and Freund's incomplete adjuvant were used to emulsify the antigen at a volume ratio of 1:1 for the initial and additional immunizations, respectively. During the breeding of laying hens, they were allowed to eat and drink freely. The room temperature was 24±2℃, the relative humidity was 70±10%, the light duration was 12-16h, and the light intensity was 16Lux. The eggs after three immunizations were collected and stored at 4℃ for the extraction and purification of IgY and the determination of antibody titer.

[0042] Extract yolk antibodies from yolk fluid using polyethylene glycol precipitation method. Collect fresh eggs after three immunizations, wash the dirt on the surface of the eggshell with clean water, soak it in a chlorhexidine solution for 1-3 minutes, wipe it dry with gauze, and then wipe it again with 75% alcohol to sterilize it. Collect the yolk under sterile conditions as much as possible. Mix the yolk fluid and pH 7.4 PBS buffer at a ratio of 1:2 (v:v). Add 3.5% (w:v) polyethylene glycol 6000 (PEG-6000), shake it on a shaker at room temperature for 20 minutes, then centrifuge it at 12,000rpm and 4℃ for 20 minutes, pour the upper liquid into two layers of filter paper for filtration, and discard the precipitate. According to the volume of the filtrate, add 8.5% PEG-6000, shake it on a shaker for 20 minutes, and then centrifuge it. Discard the supernatant, dissolve the precipitate in 10 mL of PBS buffer, add 12% PEG-6000, and centrifuge on a shaker at room temperature for 10 minutes. Discard the supernatant, dissolve the precipitate in 1.2 mL of PBS buffer, and transfer it to a dialysis bag. Dialyze it in a PBS solution at 4°C overnight, changing the dialysis solution 2 to 3 times in the middle. The next day, take out the egg yolk antibody solution from the dialysis bag, determine the egg yolk antibody protein concentration with a BCA protein quantification kit, and determine the titer of the egg yolk antibody using an indirect enzyme-linked immunosorbent assay (ELISA). After diluting the antibody to 1 mg / mL, dilute it step by step according to different concentration gradients, and determine the OD 450nm The absorbance value, when (positive OD value - blank OD value) / (control OD value - blank OD value) ≥ 2.1, the highest dilution factor is the titer of the egg yolk antibody. The remaining purified egg yolk antibody solution is stored at -20℃.

[0043] (2) Preparation of chitosan nanoparticles (CNs)

[0044] 2% chitosan (w / v) was dissolved in 0.1M acetic acid (2%, v / v), followed by the addition of 1% Tween 80 (v / v), and 10% sodium sulfate (w / v) was added to the chitosan solution, and ultrasonication was performed for 2s and 2s, respectively, for a total of 50min. Finally, the ultrasonication solution was centrifuged at 12000-14000rpm / min for 10min to obtain CNs. CNs were resuspended and washed three times with sterile ultrapure water. After the last wash, the precipitate was transferred to a centrifuge tube, 1mL was taken out for calculation of CNs concentration, and the rest was stored at 4°C for subsequent experiments.

[0045] (3) Coupling of IgY and CNs

[0046] CNs were dissolved in 0.1M sodium acetate buffer (pH 5.5) to prepare a final concentration of 1% CNs solution (w / v). IgY antibody was added to the CNs solution at a ratio of 10:2 (IgY: CNs (w / w) = 10:2). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 0.5mM) and sulfo-N-hydroxysulfosuccinimide (sulfo-NHS, 0.25mM) were directly added to the mixture as cross-linking agents and stirred at 4°C for 24h. The mixture was centrifuged at 39800×g for 10min and washed three times with sterile ultrapure water to obtain conjugated IgY-CNs. The conjugate concentration was determined by vacuum freeze drying, and the rest was stored at -20°C for subsequent experiments.

[0047] (4) Characterization and identification of IgY-CNs conjugates

[0048] Appropriate amounts of IgY, CNs, and IgY-CNs samples were weighed, freeze-dried, and made into granules with potassium bromide (1% w / w to KBr). The coupling efficiency of IgY and CNs was determined by Fourier transform infrared spectroscopy (FTIR). The spectra were collected using a ThermoScientific Nicolet 6700 FTIR spectrometer instrument, and the test spectra were between 400 and 4000 cm -1 The resolution is 4 cm, obtained by 64 scans. -1 .like Figure 1 As shown, the absorbance values ​​of CNs and IgY at 1630-1690 and 3310-3350 nm after coupling treatment were significantly higher than those of CNs, indicating that the coupling was successful.

[0049] Example 2 Evaluation of biological toxicity and anti-inflammatory activity of IgY-CNs on cells

[0050] (1) Cells and culture conditions

[0051] IPEC-J2 cells were cultured in high-glucose medium (DMEM, Solarbio) containing 10% fetal bovine serum (FBS, Gibco). The cells were cultured in an incubator at 37°C with 5% carbon dioxide until the confluence reached 85% or more. After digestion with 0.25% trypsin (Solarbio), the cells were collected and plated at 1×10 4 / well inoculated into 96-well cell culture plates, or at 1×10 6 The cells were inoculated into each well of a 6-well cell culture plate and placed in a 5% CO2, 37°C cell culture incubator for culture and adhesion.

[0052] (2) CCK-8 assay for cell viability

[0053] Cell viability was determined using the CCK8 assay. 4 / well inoculated into 96-well cell culture plates, placed in a 5% CO2, 37°C incubator for culture and adherence. Treat cells with 0, 0.25, 0.5, 1, 2, 4×MIC concentrations (i.e., 0, 0.5, 1, 2, 4, 8 mg / mL) of IgY-CNs for 24 h, then incubate with 10 μL CCK-8 solution at 37°C for 3 h, and monitor absorbance at 450 nm using an ELISA reader. Calculate cell viability using the following formula:

[0054] Cell activity / %=(A experimental well 490-A experimental well 570) / (A control well 490-A control well 570).

[0055] (3) Effects of IgY-CNs on LPS-induced cytotoxicity and inflammatory injury in IPEC-J2 cells

[0056] The cells were pretreated with IgY-CNs concentration (0, 1, 2, 4 mg / mL) for 24 hours, and then LPS 10.0 μg / mL was added for induction for 12 hours. The experiments were grouped as follows: Control: IPEC-J2 cells were cultured only without treatment; LPS: When the cell fusion reached more than 85%, the cells were induced with a medium containing 10 μg / mL LPS for 12 hours; 1 mg / mL+LPS, 2 mg / mL+LPS and 4 mg / mL+LPS groups: When the cell fusion reached more than 85%, the cells were pre-cultured with a medium containing 1, 2 or 4 mg / mL of anti-resistant Escherichia coli IgY-CNs for 24 hours, and then induced with a medium containing 10 μg / mL LPS for 12 hours, and the cell survival rate was determined using a CCK-8 kit.

[0057] After the cells were grouped and treated as above, the expression levels of genes related to inflammatory factors (IL-8, IL-10, TNF-α), TLR4 / NF-κB signaling pathway and tight junction-related genes (Occludin, Claudin-2, ZO-1) were measured.

[0058] Depend on Figure 2It can be seen that compared with the control group, LPS stimulation significantly increased the relative mRNA expression of IL-8, TNF-α, TLR4, MyD88, and NF-κB in IPEC-J2 cells, and significantly decreased the relative mRNA expression of IL-10 (P<0.05). The relative mRNA expression of IL-8 in the 2 and 4 mg / mL IgY-CNs pretreatment groups was significantly lower than that in the LPS group (P<0.05). 1, 2, and 4 mg / mL IgY-CNs pretreatment could significantly alleviate the increase in the relative mRNA expression of TNF-α, MyD88, and NF-κB in cells caused by LPS attack (P<0.05). The relative mRNA expression of IL-10 in the 2 and 4 mg / mL IgY-CNs pretreatment groups was significantly higher than that in the LPS group (P<0.05), and there was no significant change in the relative mRNA expression of TLR4. However, the relative expression of TLR4 mRNA in cells pretreated with 1 mg / mL IgY-CNs was significantly lower than that in the LPS group (P < 0.05). This indicates that cells pretreated with IgY-CNs can effectively alleviate the cellular inflammatory response caused by LPS. In a comprehensive comparison, cells pretreated with 2 mg / mL IgY-CNs had the best alleviating effect. Therefore, IgY-CNs can regulate inflammatory factors and alleviate the inflammatory response of IPEC-J2 cells induced by LPS.

[0059] Depend on Figure 3 It can be seen that compared with the control group, LPS stimulation significantly reduced the relative mRNA expression of ZO-1, claudin-2, and occludin in IPEC-J2 cells. The relative mRNA expression of ZO-1, claudin-2, and occludin in the 1, 2, and 4 mg / mL IgY-CNs pretreatment groups was significantly increased and higher than that in the control group. In addition, the 2 mg / mL IgY-CNs pretreatment group had the best effect in alleviating LPS-induced cell damage. Therefore, IgY-CNs can enhance the intestinal barrier and alleviate LPS-induced inflammatory damage in IPEC-J2 cells.

[0060] According to the results of the previous study, adding 2 mg / mL of IgY-CNs has the best effect on relieving the effects of LPS-induced IPEC-J2 cells without causing cytotoxicity. The optimal dose treatment group was selected for subsequent experiments. Figure 4As shown in Table 1, compared with the control group, after LPS stimulation of IPEC-J2 cells, the protein expression of phosphorylated p-P65 and TLR4 increased significantly (P<0.05), and the protein expression of P65 did not change significantly (P>0.05), indicating that LPS induced the activation of the NF-κB pathway and triggered an inflammatory response. Compared with the LPS group, 2 mg / mL IgY-CNs significantly reduced the protein expression of p-P65 and TLR4 in IPEC-J2 cells (P<0.05). The results showed that IgY-CNs may have a relieving effect on the inflammatory response by inhibiting the activation of the NF-κB pathway and reducing the release of inflammatory factors.

[0061] Table 1 Effect of IgY-CNs on protein expression of IPEC-J2 cells under LPS stimulation

[0062]

[0063] Example 3 Effect of IgY-CNs on the intestinal microflora of mice

[0064] (1) Test materials

[0065] Forty healthy female SPF Kunming mice aged 4 weeks with an average body weight (BW) of about 23.35±0.24g were selected for the experiment. The mice were kept at a temperature of 22-25℃ under natural light, and were fed with free drinking water and food. The experimental animals and feeds were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd. Three days before the start of the experiment, the mice were pre-fed and weighed after fasting for 12h. The 40 mice were randomly divided into four groups according to their body weight, namely the control group, low-dose and medium-dose IgY-CNs groups, with 10 mice in each group, and the experimental period was 10 days. After the start of the experiment, the mice were gavaged according to their initial body weight (IBW). The control group was gavaged with 0.3mL / mouse of physiological saline every day, and the low-dose (0.5g / kg IBW) and medium-dose (1g / kg IBW) IgY-chitosan nanoparticle groups were gavaged with different concentrations of IgY-chitosan nanoparticles, and the gavage volume was 0.3mL / mouse.

[0066] (2) Sample collection

[0067] After the experiment, the mice were fasted for 12 hours, weighed, and slaughtered. The cecal contents of the mice were collected in cryopreservation tubes, quickly frozen with liquid nitrogen, and stored in a -80°C refrigerator for the determination of intestinal microbial indicators.

[0068] (3) Measurement indicators and methods

[0069] The mouse cecal content samples were thawed, and the genomic DNA of cecal chyme bacteria was extracted using a commercial fecal DNA extraction kit. The DNA purity, concentration, and integrity were detected by NanoDrop2000 and 1% agarose gel electrophoresis. The extracted genomic DNA was used as a template to amplify the V3-V4 region of the bacterial 16S rRNA gene by polymerase chain reaction (PCR), and sequenced on the platform by Meiji Bio. The taxonomic composition and abundance were visualized by MEGAN and GraPhlAn. Based on the Kruskal-Wallis and Wilcoxon tests, the linear discriminant analysis effect size (LEfSe) was used to detect the differential abundance among different groups.

[0070] (4) Data analysis

[0071] All data were summarized and sorted by Excel, and SPSS26.0 analysis software was used to perform one-way analysis of variance (ANOVA) to compare the differences between groups, and Duncan's method was used for multiple comparisons. GraphPad Prism9 software was used to draw the graph. The data of each group were expressed as "mean ± standard error", P < 0.01 indicated an extremely significant difference, and P < 0.05 indicated a significant difference.

[0072] (5) Results and analysis

[0073] ① Community composition analysis

[0074] Door level: By Figure 5It can be seen that the top ten bacterial groups in the cecal contents of mice are: Firmicutes, Desulfobacterota, Actinobacteriota, Bacteroidota, Patescibacteria, Verrucomicrobiota, Campilobacterota, Proteobacteria, Cyanobacteria, and Deferribacterota. Compared with the control group, the relative abundance of Firmicutes in the low-dose group decreased by 5.91%, while that in the medium-dose group increased by 6.02%; the relative abundance of Desulfobacterota in the low-dose group increased by 12.9%, while that in the medium-dose group increased by 4.24%; the relative abundance of Actinobacteriota in the low-dose group decreased by 3.97%, while that in the medium-dose group decreased by 6.23%; the relative abundance of Bacteroidota in the low-dose group decreased by 7.26%, while that in the medium-dose group decreased by 2.98%.

[0075] Genus level: Figure 6 It can be seen that the dominant bacterial genera in the mouse cecal microorganisms are mainly Lactobacillus, Desulfovibrio, unclassified_f__Lachnospiraceae, Lachnospiraceae_NK4A136_group, Dubosiella, and Enterorhabdus. Among them, compared with the control group, the relative abundance of Lactobacillus in the low-dose group increased by 18.66%, and the medium-dose group increased by 18.71%; the relative abundance of Enterorhabdus in the low-dose group decreased by 5.82%, and the medium-dose group decreased by 6.79%. It shows that IgY-CNs can increase the relative abundance of intestinal probiotics Lactobacillus and have a potential inhibitory effect on pathogenic bacteria Enterobacter.

[0076] ②Species difference analysis

[0077] Door level: By Figure 7It can be seen that compared with the control group, the average relative abundance of Campylobacterota in the low-dose group was significantly decreased (P < 0.05); the average relative abundance of Deferribacterota in the low-dose group and the medium-dose group was extremely significantly decreased (P < 0.01).

[0078] Genus level: Figure 8 It can be seen that in the Bacteroidetes, compared with the control group, the Alloprevotella genus in the low-dose group was significantly decreased, and the medium-dose group was significantly increased (P < 0.05); the Alistipes, Rikenellaae_RC9_gut_group, and Rikenella genus in the low-dose and medium-dose groups were significantly decreased (P < 0.05), and the Odoribacter genus was extremely significantly decreased (P < 0.01).

[0079] Among Proteobacteria, compared with the control group, the Helicobacter genus in the low-dose group was significantly reduced (P < 0.05); the Bilophila genus in both the low-dose and medium-dose groups was extremely significantly reduced (P < 0.01).

[0080] Among Firmicutes, compared with the control group, the genera Oscillibacter and Enterococcus in the medium-dose group increased significantly (P < 0.05); the genera Tyzzerella in the medium-dose group increased extremely significantly (P < 0.01); the genera Anaerofustis in the low-dose and medium-dose groups increased extremely significantly (P < 0.01). The genera Mucispirillum in the low-dose and medium-dose groups decreased extremely significantly (P < 0.01).

[0081] The above results indicate that IgY-CNs may reduce the relative abundance of bacterial genera associated with intestinal inflammation or pathogenic infection and optimize the intestinal microecological balance, thereby reducing the host's inflammatory response.

[0082] ③Lefse multi-level species difference discriminant analysis

[0083] Control group and low-dose group: Fig. 9A shows that the abundance of g__Blautia, g__Bacteroides, g__Colidextribacter, g__Helicobacter, g__Odoribacter, g__Alloprevotella, g__Alistipes, g__Oscillibacter, g__Mucispirillum, g__Rikenella, and g__Rikenellaceae_RC9_gut_group in the control group was significantly higher than that in the low-dose group.

[0084] The abundance of c__Bacilli, f__Lactobacillaceae, g__Lactobacillus, o__Lactobacillales, g__Desulfovibrio, c__Verrucomicrobiae, g__Akkermansia, f__Akkermansiaceae, p__Verrucomicrobiota, o__Verrucomicrobiales, Anaerofustis, and o__Eubacteriales in the low-dose group were significantly higher than those in the control group. Among them, the genera of Bacilli, Lactobacillaceae, Lactobacillus, and Lactobacillales are mainly related to lactic acid fermentation, which helps maintain the acidic environment of the intestine and inhibits the growth of harmful bacteria. Verrucomicrobiae, Akkermansia, Akkermansiaceae, Verrucomicrobiota, and Verrucomicrobiales: These genera are related to the health of the intestinal mucosa and immune regulation, especially Akkermansia, which is believed to improve the intestinal barrier function and have anti-inflammatory effects. Faecalibaculum is related to intestinal health and may be involved in the degradation of polysaccharides and the production of short-chain fatty acids. The results showed that low-dose IgY-CNs had a positive effect on the intestinal microecology of mice, such as promoting lactic acid fermentation, improving intestinal mucosal health and immune regulation.

[0085] Control group and medium dose group: Fig. 9 B shows that the abundance of g__Odoribacter, g__Alistipes, g__Mucispirillum, g__Rikenella, g__Bilophila, and g__Rikenellaceae_RC9_gut_group in the control group was significantly higher than that in the medium-dose group. The abundance of g__Enterococcus and g__Tyzzerella in the medium-dose group was significantly higher than that in the control group.

[0086] Low-dose group and medium-dose group: Fig. 9 C shows that the abundance of g_Faecalibaculum in the low-dose group was significantly higher than that in the medium-dose group.

[0087] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A conjugate, which is formed by covalently coupling egg yolk antibody and chitosan through amide bonds formed by carboxyl groups and amino groups.

2. The conjugate according to claim 1, wherein the chitosan is nano-chitosan; And / or, the egg yolk antibody is a specific egg yolk antibody specific to the drug-resistant Escherichia coli antigen; And / or, the nano-chitosan and the egg yolk antibody form a covalent coupling under the action of cross-linking agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS); And / or, the egg yolk antibody and nano-chitosan are mixed in a sodium acetate buffer at a weight ratio of 5-10:1-3 for covalent coupling; And / or, the coupling is reacted at 0-8°C for 10-30 hours; And / or, the conjugate is freeze-dried to prepare nanoparticles.

3. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 2, and a pharmaceutically acceptable excipient.

4. A feed additive, comprising the conjugate according to any one of claims 1 to 2 and acceptable excipients.

5. The pharmaceutical composition according to claim 3 or the feed additive according to claim 4, for use in humans or animals, especially in livestock animals.

6. Use of the conjugate according to any one of claims 1 to 2 for preparing a medicament with anti-inflammatory activity for use in humans or animals.

7. The use according to claim 6, wherein the anti-inflammatory activity is to inhibit or alleviate the decrease in IPEC-J2 cell proliferation activity caused by inflammatory response; Preferably, the anti-inflammatory activity is to reduce the inflammatory response of IPEC-J2 cells through the NF-κB signaling pathway, and / or the anti-inflammatory activity is to alleviate the LPS-induced inflammatory damage of IPEC-J2 cells by regulating the secretion of inflammatory cytokines IL-6, IL-8, IL-10, and TNF-α.

8. Use of the conjugate according to any one of claims 1 to 2 for preparing a drug or feed additive for regulating the intestinal microbial ecology of humans or animals.

9. The use according to claim 8, wherein regulating intestinal microbial ecology refers to promoting lactic acid fermentation, improving intestinal mucosal health and / or immune regulation; Preferably, the intestinal microbial ecology is regulated by increasing the abundance of Bacillus, Lactobacillaceae, Lactobacillus, Lactobacillales, Verrucomicrobiae, Akkermansia, Akkermansiaceae, Verrucomicrobiota, Verrucomicrobiales, and Faecalibaculum, thereby improving the health of the intestinal mucosa, improving intestinal barrier function, and immune regulation.

10. The use according to any one of claims 6 to 9, wherein the animal body is pig, sheep, cattle, chicken, especially piglet.

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