Nocardiosis-resistant egg yolk antibody, and preparation method and application thereof

Abstract

The application discloses an egg yolk antibody against Nocardiosis and a preparation method and application thereof, and relates to the technical field of biological medicines. A light chain sequence of the egg yolk antibody comprises a sequence shown in SEQ ID NO. 1, and a heavy chain sequence comprises a sequence shown in SEQ ID NO. 2. The application expands culture of pathogenic bacteria Nocardia, prepares an inactivated bacterin vaccine, and immunizes hens, so that the hens produce egg yolk antibodies against Nocardia through an immune response. After immunization, the application collects eggs, extracts the egg yolk antibodies against Nocardiosis from egg yolk by using a water dilution method. It is verified that the egg yolk antibodies have a strong binding capacity with Nocardia, can effectively inhibit Nocardia, and the titer can reach 1:64000 at the highest.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an egg yolk antibody against Nocardiosis and a preparation method and application thereof. BACKGROUND

[0002] Nocardiosis is a bacterial disease caused by Nocardia seriolae infection in fish, which is mainly characterized by body surface bleeding and ulceration, and a large number of white nodules in the body. The pathogenic bacteria Nocardia seriolae is a gram-positive bacteria, which mainly lives as saprophyte and is widely distributed. Nocardiosis is prone to occur in seasons with higher temperature such as summer. The incubation period of the disease is long, and there are no obvious symptoms at the early stage, so it is difficult to diagnose and treat in the early stage. The mortality rate of the disease is high, and the disease course is long. Once it breaks out, it often causes serious economic losses. The diseased fish generally shows symptoms of body surface bleeding and ulceration, and the liver, kidney, spleen and other organs can be seen a large number of yellow-white nodules, and a large number of nodules can also be seen in the muscle tissue of the body. With the aggravation of the disease, symptoms such as red and swollen anus and abdominal distension may occur (as shown in Figure 1

[0003] At present, in the process of fish breeding, the treatment of Nocardiosis often adopts the treatment mode of improving water quality, taking chemical antibacterial drugs such as sulfonamide antibacterial drugs internally and using disinfectant drugs externally. The common problem of using antibacterial drugs for treatment is that drug-resistant strains are easy to produce. At present, research has indeed found drug-resistant strains, which will greatly reduce the treatment effect and also have certain adverse consequences on environmental safety. Therefore, it is urgent to find green, safe and environmentally friendly alternative drugs that will not produce drug resistance.

[0004] Egg yolk antibody (egg yolk immunoglobulin Y, IgY) is a green and pollution-free drug with high specificity, no drug resistance and no residue, which is an ideal substitute for chemical antibacterial drugs, and therefore has attracted extensive attention and research. Chicken egg yolk antibody is produced by immune response of hens after being stimulated by antigens, and is stored in egg yolk, hence the name. IgY has many advantages. In production, hens only need a small amount of antigen for immunization to produce a large amount of target antibody. Because IgY is stored in egg yolk, it only needs to collect eggs for extraction, without the need for blood sampling and other operations that harm hens, reducing harm to experimental animals and protecting animal welfare. In application, IgY has good stability, strong acid and alkali resistance, and is not greatly affected after repeated freezing and thawing. It has certain resistance to digestive enzymes in the digestive tract, and even after being enzymatically digested into small fragments, it can still play a passive immunization role to protect susceptible animals. In addition, due to the distant genetic relationship, the specificity and sensitivity of IgY are better than those of IgG, and IgY does not activate the complement system, which can effectively reduce the occurrence of inflammatory reactions.

[0005] ​Because IgY has the ability to specifically bind to corresponding antigens, it is highly targeted to the corresponding diseases. Currently, there are many studies on its use in the prevention and treatment of viral and bacterial animal diseases. IgY's many advantages make it a promising candidate for animal disease diagnosis, prevention and treatment. Summary of the Invention

[0006] The purpose of this invention is to provide an egg yolk antibody against Nocardiac disease, its preparation method, and its application, in order to solve the problems existing in the prior art. The egg yolk antibody provided by this invention has a strong binding ability with Nocardia and can effectively inhibit Nocardia.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides an egg yolk antibody against nocardiosis, wherein the light chain sequence of the egg yolk antibody includes the sequence shown in SEQ ID NO.1, and the heavy chain sequence includes the sequence shown in SEQ ID NO.2.

[0009] The present invention also provides the use of the above-described egg yolk antibody in the preparation of medicaments for the prevention and / or treatment of nocardiosis.

[0010] The present invention also provides a medicament for the prevention and / or treatment of nocardiosis, the active ingredient of which includes the above-mentioned egg yolk antibody.

[0011] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0012] The present invention also provides the application of the above-mentioned egg yolk antibody in the preparation of a kit for detecting Nocardia.

[0013] The present invention also provides a kit for detecting nocardiosis, comprising the above-mentioned egg yolk antibody.

[0014] Furthermore, the kit also includes an enzyme-labeled plate, blocking solution, washing solution, and colorimetric reagent.

[0015] The present invention also provides a method for preparing the above-mentioned egg yolk antibody, characterized by comprising the following steps:

[0016] (1) Nocardia was inactivated to obtain inactivated bacteria;

[0017] (2) Immunize laying hens with the inactivated bacteria and collect eggs;

[0018] (3) The egg yolk antibody was extracted from the egg.

[0019] Furthermore, in step (1), the Nocardia bacteria are inactivated using formaldehyde.

[0020] Furthermore, in step (3), the extraction is performed using a water dilution-salting-out method.

[0021] The present invention discloses the following technical effects:

[0022] This invention involves expanding the culture of the pathogenic bacterium Nocardia to produce an inactivated vaccine for immunizing laying hens, thereby inducing an immune response and the production of anti-Nocardia IgY. Immunized eggs are collected, and anti-Nocardia IgY is extracted from the yolk using a water dilution-salting-out method. Indirect ELISA is used to detect antibody titers at different dates, and antibody titer curves are plotted to analyze titer trends. Immunofluorescence is used to detect the specificity of anti-Nocardia IgY, with non-specific IgY as a control, to assess the binding strength of anti-Nocardia IgY to the pathogen. Subsequently, the in vitro antibacterial effect of anti-Nocardia IgY is tested using the drug sensitivity test, measuring the size of the inhibition zone and examining the antibacterial effects of different concentrations of anti-Nocardia IgY at different bacterial concentrations and time periods. This provides a theoretical basis for further exploring the mechanism of action of anti-Nocardia IgY in providing passive immune protection to diseased fish and for the prevention and control of Nocardiac disease. The results showed that the highest IgY titer after immunization reached 1:64000, and this level could be maintained for 40–50 days. Immunofluorescence and drug susceptibility testing were used to detect the in vitro antibacterial activity of anti-Nocardia IgY. The results showed that compared to non-specific IgY, anti-Nocardia IgY had a strong binding affinity to Nocardia, and its in vitro antibacterial effect was concentration-dependent, reaching its optimal effect at 64 hours. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Dissection and observation of diseased largemouth bass; where A: body surface; B: ventral side; C: internal tissues; D: organs;

[0025] Figure 2 To determine the optimal antigen coating concentration using the checkerboard method;

[0026] Figure 3 The trend of changes in the titer of anti-Nocardia specific IgY after immunization is shown, with arrows indicating immunization time;

[0027] Figure 4 The results are SDS-PAGE assays for specific IgY; where M: Marker; 1: IgY extract; 2: IgY precipitate 1; 3: IgY precipitate 2.

[0028] Figure 5 The results of SDS-PAGE silver staining for specific IgY are shown; where M: protein marker; 1: IgY extraction solution; 2: first precipitated IgY.

[0029] Figure 6 This study describes the immunofluorescence assay for the binding of specific IgY to Nocardia. A represents specific IgY with Nocardia; B represents non-specific IgY with Nocardia; C represents PBS with Nocardia; D, E, and F represent the results of A, B, and C observed under bright field conditions, respectively.

[0030] Figure 7 The antibacterial effects of different concentrations of bacterial solutions;

[0031] Figure 8 The antibacterial effects of different concentrations of antibodies were observed. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1

[0038] 1. Materials and Methods

[0039] 1.1 Laboratory animals and pathogens

[0040] Healthy laying hens aged 110–120 days were purchased from Hongxing Poultry and Egg Store in Fenhu Town, Wujiang District, Suzhou City.

[0041] Nocardia seriolae was purchased from Mingzhou Biotechnology.

[0042] 1.2 Reagents and their preparation

[0043] (1) PBS buffer (0.01M, pH 7.4): NaCl 8.00g, KCl 0.20g, Na2HPO4 1.44g, KH2PO4 0.24g, deionized water to a final volume of 1L, sterilize, and store at 4℃.

[0044] (2) LB liquid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, and deionized water to a final volume of 1000mL, then sterilize. For solid medium, add 15g agar powder.

[0045] (3) BHI liquid medium: 10g peptone, 12.5g dehydrated calf brain extract powder, 5g dehydrated calf heart extract powder, 5g sodium chloride, 2g glucose, 2.5g disodium hydrogen phosphate, and deionized water to a final volume of 1000mL. Sterilize. For solid medium, add 15g agar powder.

[0046] (4) Adjuvant: 803 mL of span and 1 g of aluminum stearate were dissolved in white oil, heated until no bubbles were observed, and the volume was adjusted to 50 mL. The solution was then poured into a blue sprue bottle and sterilized at 121 °C for 30 min. (The white oil was domestically produced vaccine-grade white oil).

[0047] (5) TBS: Tris 12.1g, NaCl 17.5g, bring to a final volume of 2L, and adjust the pH to 7.4 with concentrated hydrochloric acid.

[0048] (6) T-TBS: 0.5‰ of Tween-20 is added to TBS.

[0049] (7) Coating solution: NaHCO3 2.93 g, Na2CO3 1.59 g; bring the volume up to 1000 mL, and adjust the pH to 9.6 with NaOH.

[0050] (8) Sealing liquid: 10% skim milk powder.

[0051] (9) Colorimetric solution A: 0.96g citric acid, dissolved in single-distilled water, and diluted to 50mL. Colorimetric solution B: 3.585g disodium hydrogen phosphate, dissolved in single-distilled water, and diluted to 50mL. (Colorimetric solution: 15.1mL A solution + 16.9mL B solution + 12mg OPD + 120μL 30% H2O2, prepare fresh and protect from light.)

[0052] (10) Termination solution: 10 mL of 98% concentrated sulfuric acid was diluted to 100 mL of single-distilled water.

[0053] (11) 0.12 mol / L acetic acid-sodium acetate buffer: 9.84 g sodium acetate, diluted to 1 L with deionized water, and pH adjusted to 5.1 with acetic acid.

[0054] (12) Staining solution: 225 mL methanol, 50 mL glacial acetic acid, 225 mL deionized water, 1.25 g Coomassie brilliant blue.

[0055] (13) Decolorizing solution: 450mL methanol, 100mL glacial acetic acid, and deionized water to a final volume of 1L.

[0056] (14) Fixative: 20 mL methanol, 5 mL glacial acetic acid, 25 mL ddH2O.

[0057] (15) Rinse solution: ddH2O.

[0058] (16) Sensitizing solution: 15 mL methanol, 3.4 g anhydrous sodium acetate, 0.1 g sodium thiosulfate, 35 mL ddH2O.

[0059] (17) Silver staining solution: 0.125g silver nitrate, 50mL ddH2O.

[0060] (18) Colorimetric solution: 1.25g anhydrous sodium carbonate, 20μL formaldehyde, 50mL ddH2O.

[0061] (19) Termination solution: 0.2g glycine, 50mL ddH2O.

[0062] (20) Specific IgY: Extracted and purified from immunized eggs.

[0063] (21) Non-specific IgY: It was extracted and purified from unimmunized eggs.

[0064] (22) Other reagents: 75% alcohol, glycerol, ddH2O, formaldehyde, PBS containing 4% Tween, BCA kit purchased from Solarbio, HRP goat anti-chicken IgY purchased from Sigma.

[0065] 1.3 Preparation of inactivated bacterial vaccines

[0066] Nocardia bacteria stored at -80℃ were inoculated into BHI medium and cultured at 28℃ with shaking for 72 h. The bacterial cells were collected by centrifugation at 6000 rpm for 30 min and resuspended in 0.01 mol / mL phosphate buffer. 1% formaldehyde was added, and the culture was incubated at 28℃ with shaking for 48 h. 0.1 mL of the inactivated bacterial suspension was spread onto BHI plates and incubated at 28℃ for 72 h to test the inactivation effect. The inactivated bacterial suspension was centrifuged at 6000 rpm for 30 min, washed twice with PBS, and resuspended in PBS containing 4 wt% Tween to adjust the bacterial concentration to 1 × 10⁻⁶. 9 CFU / mL, store at -20℃.

[0067] 1.4 Immunization of laying hens

[0068] Mix the inactivated vaccine and adjuvant at a volume ratio of 1:1 until homogeneous, forming a water-in-oil emulsion. Inject 1 mL per chicken subcutaneously into the left and right pectoral muscles and neck. Immunize three times using the same dosage and method, with a second immunization 10 days after the first, and a third immunization 20 days after the second.

[0069] 1.5 Egg collection and antibody treatment

[0070] Eggs were collected periodically after immunization, their surfaces were cleaned, and they were numbered and stored. For testing, the egg whites and yolks were mixed thoroughly using a food processor. 50 mg of the mixed egg liquid was weighed and added to 100 mL of 1 wt% skim milk powder, then vortexed to mix thoroughly (2000-fold dilution). 50 mg of unimmunized egg liquid was weighed and dissolved in 100 mL of 1 wt% skim milk powder, then vortexed to mix thoroughly (2000-fold dilution) as a control.

[0071] 1.6 Enzyme-linked immunosorbent assay (ELISA) for antibody titer detection

[0072] Before using indirect ELISA to detect antibody titers in egg liquid, a checkerboard ELISA method is first used to screen for a suitable antigen coating concentration to obtain the most suitable detection conditions.

[0073] 1.6.1 Using the checkerboard method to screen for suitable antigen concentrations

[0074] (1) Coating: Dilute inactivated Nocardia bacteria with carbonate buffer to 1×10⁻⁶. 9 1×10 8 1×10 7 1×10 6 1×10 5 1×10 4 1×10 3 CFU / mL, one concentration gradient per row, coated a 96-well microplate, add 100 μL to each well, incubate at 37°C for 1 h, and then at 4°C overnight.

[0075] (2) Washing: Wash with PBS for 5 min, 300 μL per well, repeat 3 times.

[0076] (3) Blocking: 300 μL of 10 wt% skim milk powder in PBS per well, incubated at 37°C for 2 h.

[0077] (4) Washing: Same as (2).

[0078] (5) Incubation of primary antibody: Egg liquid was added to each column at dilutions of 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000 using 1 wt% skim milk powder. The last column was the normal control. The last row of primary antibody was incubated with TBS as a blank control. The dilution was repeated in two wells, 100 μL per well, and incubated at 37°C for 2 h.

[0079] (6) Washing: Wash with PBST for 5 min, 300 μL per well, repeat 4 times; wash with PBS for 5 min, 300 μL per well.

[0080] (7) Incubation of secondary antibody: 1 wt% skim milk powder was diluted with HRP goat anti-chicken IgY (purchased from Sigma) at a ratio of 1:2000. 100 μL was added to each well and incubated at 37°C for 1 h.

[0081] (8) Washing: Same as 6.

[0082] (9) Color development: Add 100 μL of color developer to each well and react for 5 min.

[0083] (10) Termination: Add 50 μL of 2M H2SO4 to each well.

[0084] (11) Plate reading: OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 490 value.

[0085] 1.6.2 Indirect ELISA method for detecting antibody titer

[0086] (1) Coating: Dilute inactivated Nocardia bacteria with carbonate buffer to 1×10⁻⁶. 7 CFU / mL, coat the microplate, add 100 μL to each well, incubate at 37°C for 1 h, then at 4°C overnight.

[0087] (2) Washing: Wash with TBS for 5 min, 300 μL per well, repeat 3 times.

[0088] (3) Blocking: 300 μL of 10 wt% skim milk powder in PBS per well, 37°C, wet incubation for 1.5 h.

[0089] (4) Washing: Same as 2.

[0090] (5) Incubation of primary antibody: Add the treated egg solution to each column at dilutions of 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000. The last column is the normal control. The last row is the blank control. Each dilution is repeated in two wells, with 100 μL per well. Incubate at 37°C for 2 hours.

[0091] (6) Washing: Wash with T-TBS for 5 min, 300 μL per well, repeat 5 times; wash with TBS for 5 min, 300 μL per well, repeat 2 times.

[0092] (7) Incubation of secondary antibody: 1 wt% skim milk powder was diluted with HRP goat anti-chicken IgY (purchased from Sigma) at a ratio of 1:2000. 100 μL was added to each well and incubated at 37°C for 1 h.

[0093] (8) Washing: Same as 6.

[0094] (9) Color development: Add 100 μL of color developer to each well and react for 5 min.

[0095] (10) Termination: Add 50 μL of 2M H2SO4 to each well.

[0096] (11) Plate reading: OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 490 value.

[0097] 1.7 Extraction and purification of anti-Nocardia IgY

[0098] The extraction and purification of IgY were performed using the water dilution-salting-out method, and the specific steps are as follows:

[0099] (1) Mix egg yolk and acetate-sodium acetate buffer at a volume ratio of 1:9 on a magnetic stirrer for 10 min.

[0100] (2) Let stand overnight at 4℃.

[0101] (3) Centrifuge at 4℃ and 5000rpm for 20min and collect the supernatant.

[0102] (4) Add ammonium sulfate crystals to a final concentration of 19wt%, let stand for 1 hour, centrifuge at 12000 rpm for 10 minutes, and collect the precipitate.

[0103] (5) Dissolve the PBS in the original volume, add ammonium sulfate crystals to a final concentration of 13wt%, let stand for 2 hours, centrifuge at 12000 rpm for 10 minutes, and take the precipitate.

[0104] (6) Dialyze with PBS for 24 hours to obtain purified anti-Nocardia IgY.

[0105] (7) Sequencing of purified anti-Nocardia IgY.

[0106] 1.8 Determination of protein content in purified anti-Nocardia IgY solution using the BCA method

[0107] The protein content was determined using a BCA kit, and the steps are as follows:

[0108] (1) Mix BCA reagent and Cu reagent in a 50:1 ratio to prepare BCA working solution.

[0109] (2) Dilute BSA standard (2 mg / mL) with PBS to different concentrations: 0, 0.2, 0.4, 0.6, 0.8, 1.2, 1.6, 2 mg / mL, and add 20 μL to the microplate.

[0110] (3) Dilute the sample appropriately and add 20 μL to the microplate.

[0111] (4) Add 200 μL BCA working solution and react at 37°C for 25 minutes. Measure A using a microplate reader. 562 The protein content of the sample is calculated by plotting a curve using the values ​​of the standard.

[0112] 1.9 SDS-PAGE assay for antibody purity

[0113] 1.9.1 Polyacrylamide gel electrophoresis

[0114] (1) Dilute the precipitate with PBS (0.01M, pH 7.4).

[0115] (2) Add sample loading buffer, boil in water for 15 min, centrifuge at 4℃ and 12000 rpm for 10 min, and take the supernatant for sample loading.

[0116] (3) Prepare a 15wt% separating gel, slowly add isopropanol, let stand at 37°C for 30 min, and pour off the isopropanol; prepare a 5wt% stacking gel, insert the comb, let stand for 20 min, remove the comb, place it on an electrophoresis apparatus containing electrophoresis buffer, and remove the air bubbles.

[0117] (4) Load 3 μL of marker and 10 μL of sample respectively, electrophoresis at 80V for 30 min, and electrophoresis at 120V for 90 min.

[0118] (5) Stain the gel with staining solution for 3 hours.

[0119] (6) Decolorize with decolorizing solution and observe the protein bands.

[0120] 1.9.2 SDS-PAGE Polyacrylamide Gel – Silver Staining Method

[0121] Immerse the gel in 5 times its volume of fixative and react on a shaker for 30 minutes. Replace with an equal volume of sensitizing solution and react for another 30 minutes. Wash three times with an equal volume of water, 5 minutes each time. Replace with silver staining solution and stain in the dark for 5 minutes. Wash twice with water, 1 minute each time. Replace with developing solution and gently shake by hand until the protein bands reach the desired clarity. Add stop solution to stop the reaction.

[0122] 1.10 Immunofluorescence detection of the specificity of anti-Nocardia IgY

[0123] (1) Activate Nocardia, centrifuge at 5000 rpm for 10 min to collect the cells, and wash three times with PBS.

[0124] (2) The IgY was resuspended separately in PBS solutions containing 50 mg / mL specific IgY and non-specific IgY, with the PBS solution serving as a blank control. The final concentration for both solutions was 1.0 × 10⁻⁶. 8 Take 1 mL of each of the CFU / mL values ​​and incubate them in a 28℃ incubator for 2 hours.

[0125] (3) Collect bacterial cells by centrifugation at 5000 rpm for 10 min, wash three times with PBS, resuspend in PBS solution containing FITC-labeled goat anti-chicken IgG diluted 1:100, and incubate at 28℃ for 1 h.

[0126] (4) After labeling, wash three times with PBS, take 10 μL and drop it into a confocal glass dish, and observe it using a single-photon confocal fluorescence microscope.

[0127] 1.11 Detection of the in vitro antibacterial effect of anti-Nocardia IgY using the drug susceptibility testing method

[0128] (1) Preparation of bacterial suspension: After activating Nocardia, 50 μL was inoculated into 5 mL of BHI medium and cultured at 28°C with shaking for 5 days. The bacterial concentration was then adjusted to 1 × 10⁻⁶. 9 1×10 8 1×10 7 CFU / mL.

[0129] (2) Take 200 μL of the prepared solid BHI medium with a concentration of 1×10⁻⁶. 9 1×10 8 1×10 7 CFU / mL bacterial suspension was evenly spread on solid BHI medium, with two replicates for each concentration.

[0130] (3) Place the drug sensitivity test strips on the plate, two strips per plate, and add 40 μL of 160 mg / mL anti-Nocardia IgY and 160 mg / mL non-specific IgY to each plate.

[0131] (4) Incubate at a constant temperature of 28℃ and measure the size of the inhibition zone at each time point.

[0132] (5) Select the bacterial concentration with the most obvious antibacterial effect and spread it on the plate again. Place the drug sensitivity paper on the plate, 6 plates per plate, and add 40 μL of anti-Nocardia IgY and non-specific IgY at concentrations of 160, 80, 40 and 20 mg / mL respectively.

[0133] (6) Incubate at a constant temperature of 28℃ and measure the size of the inhibition zone at each time point.

[0134] 2. Results

[0135] 2.1 Determination of the optimal antigen coating concentration

[0136] When the antigen coating concentration increases or the antibody dilution factor decreases, more specific IgY will bind to the ELISA plate, leading to the binding of more enzyme-labeled antibody and a correspondingly higher absorbance value. The optimal absorbance value for the ELISA reader is between 1 and 2. A P / N value > 2.1 indicates a positive result. (Checkerboard ELISA results) Figure 2 The data shows that when the coating antigen is 1×10⁻⁶, the effect is as follows: 7 When Nocardiac bacteria were inactivated at CFU / mL, the antibody P / N value was >2.1 and the absorbance was around 1.5, while the absorbance of the blank control was less than 0.1, i.e., 1×10⁻⁶. 7 CFU / mL is the optimal antigen coating concentration.

[0137] 2.2 Changes in the titer of anti-Nocardia IgY after immunization

[0138] Use 1×10 7 CFU / mL inactivated Nocardia bacteria were coated onto ELISA plates. The change in anti-Nocardia IgY titer in egg solution over time was detected starting 20 days post-immunization. Results are shown below. Figure 3 The results showed that antibody titers rose rapidly 30 days after the initial immunization, reaching 1:64,000 on day 40. After a period of time, the titer began to decline, rising again after a booster immunization on day 80, and reaching 1:64,000 again on day 100. The titer of anti-Nocardia IgY was found to be as high as 1:64,000 after immunization and could be maintained for 40–50 days. Eggs with titers above 1:64,000 were selected for further experiments based on the test results.

[0139] 2.3 Determination of protein content in purified anti-Nocardia IgY solution using BCA method

[0140] As shown in Table 1, both anti-Nocardia IgY and non-specific IgY had higher protein concentrations in the first precipitation, indicating a higher yield.

[0141] Table 1. BCA Method Detection

[0142]

[0143] 2.4 SDS-PAGE assay for antibody purity

[0144] Under the action of β-mercaptoethanol, the disulfide bonds connecting the antibody heavy and light chains are broken, thus separating them into heavy and light chains after electrophoresis. Figure 4 ).like Figure 5 As shown, the protein bands are more clearly visible after silver staining. The heavy chain size is 67–70 kDa, and the light chain size is 22–30 kDa, consistent with the target size. The figure also shows that the extract obtained after dilution with water contains a significant amount of impurities. In the precipitate obtained after salting out, the target band is clear, the impurities are significantly reduced, and the purity is significantly improved.

[0145] 2.5 Sequencing Results

[0146] Light chain sequences include:

[0147] WYQQKSPGSAPVTVIYSNDKRPSNIPSRTSLSQSTSTGTLTITGVQADDEAVYFC GNEDNSIDSAIF GAG TTLTVLGQPKVAPTITLFPPSKEELNEATKATLVCLINDFYPSPV TVDWVIDGSTRSGETTAPQRQSNSQYMASSYLSLSASDWSSHETYTCR(SEQ IDNO.1).

[0148] Heavy chain sequences include:

[0149] QTPGGALSLKCVASGFTFSSNAMGWVRQAPGKGLEFVAGIGNTGRYTGYGPAVKGRAIISRDNGQSTV RLQLNNLRAEDTAIYYCAKSASGSGYGCKGWIDWWGHGAEVVVSAASPTSPPRLYPLSACCSDSAVPPAVGCLLSP SSAGGISWEGSGGTAVTGRVSGTPVKLSFVRLSPGEKRKSFVCSAAPGGALL KKEVQVCRVGPPPPVAPEVQVLHASSCTPSQSESVELLCLVTGFSPASAEVEWLVDGVGGLLVASQSPAVRSGSTYSLSSRVNVSGTDWREGKSYSCRVRHPATNTVVEDHVKGCPDGAQSCSPIQLYAIPPSPGELYISLDAKLRCLVVNLPSDSSLSVTWTREKS GNLRPDPMVLQEHFNGTYSASSAVPVSTQDWLSGERFTCTVQHEELPLPLSKSVYRNTGPTTPPLIYPFAPHPEELSLSRVTLSCLVRGFRPRDIEIRWLRDHRAVPATEFVTTAVLPEERTANGAGGDGDTFFVYSKMSVETAKWNGGTVFACMAVHEALPMR(SEQ ID NO.2).

[0150] In the above sequences, the underlined parts are variable region sequences.

[0151] 2.6 Immunofluorescence detection of the specificity of anti-Nocardia IgY

[0152] When Nocardia citrate is co-cultured with specific IgY, it exhibits green fluorescence upon binding to FITC-labeled goat anti-chicken IgG. Figure 6 (A); When co-cultured with non-specific IgY, multiple bacteria exhibit weak fluorescence when adhering together ( ). Figure 6 (B); Nocardia citrate does not show fluorescence when co-cultured with PBS. Figure 6 (C) Figure 6 Photographs D, E, and F in the image are of A, B, and C observed under bright field conditions, respectively. The experimental results show that specific IgY has a strong binding affinity to Nocardia, while non-specific IgY has a very weak binding affinity to Nocardia.

[0153] 2.7 Detection of the in vitro antibacterial effect of anti-Nocardia IgY using the drug sensitivity test method

[0154] Comparison of different bacterial concentrations (1×10⁻⁶) 9 1×10 8 1×10 7 CFU / mL, the same concentration of IgY (160 mg / mL) versus 1×10 7 The CFU / mL concentration provides the best antibacterial effect. Figure 7 When the bacterial concentration is the same (1×10⁻⁶), 7 CFU / mL), inhibition zones began to appear on the plates after 32 hours. Comparing different antibody concentrations, it was observed that the antibacterial effect increased significantly with increasing IgY concentration. Figure 8 ). Coated at a concentration of 1×10⁻⁶ μL. 7 Using CFU / mL Nocardia plates as the observation target, the inhibition zone diameter reached its maximum at 64 hours, presumably at the beginning of the logarithmic growth phase. Afterward, the inhibition zone began to shrink. The inhibitory effect stabilized after 4 days, and other bacteria began to appear. Non-specific IgY showed poor inhibitory effect, with obvious colony growth observed.

[0155] In summary, this invention uses an indirect ELISA method to detect the titer of specific antibodies. The highest detected titer reached 1:64000, and this level persisted for 40–50 days after immunization. Based on this result, eggs from suitable dates were selected for further experiments. The water dilution-salting-precipitation method was used to extract and purify anti-Nocardia IgY. The obtained IgY was detected using the BCA protein quantification method, indicating that the protein content of the first precipitate during purification was relatively high. SDS-PAGE electrophoresis showed that the molecular weights of both the light and heavy chains of IgY were consistent with the target size, and the target band was clear, suitable for further experiments.

[0156] This invention involves extracting and purifying a high-titer anti-Nocardia IgY and then conducting in vitro antibacterial experiments. Using immunofluorescence technology, with non-specific IgY as a control, the results show that the anti-Nocardia IgY has a strong ability to bind to Nocardia in vitro, while the binding ability of non-specific IgY is very weak, emitting only faint fluorescence. Simultaneously, using the drug sensitivity test, the size of the inhibition zone was measured at different time points under different concentrations of bacterial suspension and anti-Nocardia IgY. The results indicate that the in vitro antibacterial effect of anti-Nocardia IgY is concentration-dependent; that is, the antibacterial effect increases significantly with increasing IgY concentration, with the optimal antibacterial effect at 64 hours, stabilizing after 4 days, and then the appearance of other bacteria.

[0157] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An egg yolk antibody against nocardiosis, characterized in that, The light chain sequence of the egg yolk antibody includes the sequence shown in SEQ ID NO.1, and the heavy chain sequence includes the sequence shown in SEQ ID NO.

2.

2. The use of the egg yolk antibody as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of nocardiosis.

3. A medicine for the prevention and / or treatment of nocardiosis, characterized in that, The active ingredient includes the egg yolk antibody as described in claim 1.

4. The drug according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The use of the egg yolk antibody as described in claim 1 in the preparation of a kit for detecting Nocardia.

6. A kit for detecting nocardiosis, characterized in that, It contains the egg yolk antibody as described in claim 1.

7. The reagent kit according to claim 6, characterized in that, The kit also includes an enzyme-labeled plate, blocking solution, washing solution, and colorimetric reagent.

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