CN116589573A - Preparation and application of tilapia streptococcus agalactiae egg yolk antibody - Google Patents
Preparation and application of tilapia streptococcus agalactiae egg yolk antibody Download PDFInfo
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- CN116589573A CN116589573A CN202310514222.4A CN202310514222A CN116589573A CN 116589573 A CN116589573 A CN 116589573A CN 202310514222 A CN202310514222 A CN 202310514222A CN 116589573 A CN116589573 A CN 116589573A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Genetics & Genomics (AREA)
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- Veterinary Medicine (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The application discloses a preparation and application of a tilapia streptococcus agalactiae egg yolk antibody, and relates to the field of biotechnology and application, and the technical scheme is characterized by comprising the following steps of S1, preparing streptococcus agalactiae antigen and inactivating; s2: inactivation of streptococcus agalactiae antigen; s3, immunizing laying hens and collecting eggs; s4, separating and purifying the egg yolk antibody; s5, purifying the egg yolk antibody. The streptococcus agalactiae is used as antigen to immunize the laying hen, the hyperimmune egg is collected, and the yolk of the hyperimmune egg is separated and purified to obtain the streptococcus agalactiae yolk antibody. The streptococcus agalactiae egg yolk antibody has strong specificity, higher purity and stronger inhibiting effect on streptococcus agalactiae, and simultaneously has low preparation cost, convenience and simplicity, and is suitable for large-scale production.
Description
Technical Field
The application relates to the field of biotechnology and application, in particular to preparation and application of a tilapia streptococcus agalactiae egg yolk antibody.
Background
Streptococcus agalactiae (Streptococcus agalactiae), also known as group B streptococcus (Group B streptococci, GBS), is gram positive, facultative anaerobic, has a capsule, mostly spherical, paired or arranged in chain, non-motile. In 1966, researchers first separated streptococcus in the cultivation of golden bream, and in 1974 Plumb et al first separated the same streptococcus in sea fish, and researchers judged these isolates as streptococcus agalactiae by biochemical characteristics. Streptococcus agalactiae is gram-positive bacteria, can infect various aquatic animals, and is mainly characterized by symptoms such as movement disorder, protruding eyeballs, turbidity, bleeding, flaking and the like. Above 15 ℃ the host will die, so that the tilapia streptococcus agalactiae usually occurs in summer, the survival rate of the pathogenic fish is less than 5%, and huge losses are brought to the aquaculture industry.
At present, the prevention and treatment means for the diseases caused by streptococcus agalactiae mainly comprise the following steps: antibiotics and chemical drugs, chinese herbal medicines, inactivated vaccines, antibodies and the like. Antibiotics and chemical drugs are widely used, however, with the long-term abuse of antibiotics and chemical drugs, a number of drawbacks are brought about: (1) pathogenic bacteria generate drug resistance, so that the action effect of antibiotics is obviously reduced or even ineffective; (2) serious water pollution is caused, and micro ecological unbalance of the water environment is caused; (3) antibiotic residues appear in animals, which affects the food quality safety of aquatic products and indirectly endangers the health of human bodies. Therefore, a new technology and a new product which can effectively control the diseases of the aquaculture animals and can not bring toxic and side effects are urgently needed, and the existing antibiotics or chemical medicines are partially or completely replaced, so that healthy aquaculture is realized, and the technology has become a hot spot and a difficult point of current research.
The yolk antibody (egg yolk immunoglobulin, igY) has the advantages of strong specificity, high efficiency, no toxic or side effect, no pollution to the environment and the like, and has become an effective substitute for antibiotics and chemical medicines. The yolk antibody has the characteristics of low cost, high benefit, convenient production, high yield, simple preparation and the like, and is widely paid attention to as an effective method for preventing and controlling diseases. Compared with the traditional antibiotics and chemical drugs, the application of the egg yolk antibody has the following advantages: (1) the specificity is strong: unlike broad-spectrum antibiotics, egg yolk antibodies are only directed against the corresponding pathogenic bacteria and do not affect other flora. (2) The acquisition is simple: the egg yolk antibody can be obtained from the eggs of immunized laying hens without killing a large number of animals, and the produced antibody has high titer and strong stability. (3) There is a certain phylogenetic distance between the birds and mammals and a small amount of antigen can elicit an effective immune response. (4) No residue: the yolk antibody belongs to dry immunoglobulin and can be digested and absorbed by organisms as a nutrient substance after acting. (5) Short research and development period, low cost, long storage period and convenient transportation and application. The egg yolk antibody is widely applied to the aspects of prevention and treatment of animal diseases, disease diagnosis and the like, and the American Food and Drug Administration (FDA) classifies the egg yolk antibody into 'safe use substances (Generally Recognized As Safe, GRAS)', and the specific egg yolk antibody plays roles in protecting and preventing aquatic animals and treating diseases in feeding, injection, dipping and other modes, and can also be applied to the fields of immunological detection, aquatic product preservation, disease diagnosis and the like.
Currently, patents disclosed and granted in aquatic application include anti-macrobrachium rosenbergii field virus, anti-cryptosporidiosis, anti-vibrio parahaemolyticus, anti-aeromonas hydrophila, anti-vibrio cholerae, anti-vibrio lautus and anti-vibrio anguillarum egg yolk antibodies and the like, and lack of preparation of anti-streptococcus agalactiae egg yolk antibodies.
Disclosure of Invention
The application aims to provide the preparation and application of the tilapia streptococcus agalactiae egg yolk antibody, and the prepared anti-streptococcus agalactiae egg yolk antibody has strong specificity, higher purity and stronger inhibiting effect on streptococcus agalactiae, and meanwhile, the preparation cost of the antibody is low, and the antibody is convenient and simple and is suitable for large-scale production.
The technical aim of the application is realized by the following technical scheme: the preparation and application of the tilapia streptococcus agalactiae egg yolk antibody comprise the following steps:
s1, preparing streptococcus agalactiae antigen and inactivating; inoculating streptococcus agalactiae to a BHI solid agar culture medium, culturing for about 12 hours at 37 ℃ in an inversion way, selecting single bacterial colony, inoculating to the BHI liquid culture medium, shaking and culturing overnight at 37 ℃ and 160rpm, obtaining bacterial precipitate and supernatant containing streptococcus agalactiae through centrifugal separation, and washing bacterial precipitate for 3 times by using PBS buffer solution to obtain streptococcus agalactiae suspension;
s2: inactivation of streptococcus agalactiae antigen; adding 4% paraformaldehyde fixing solution into the streptococcus agalactiae suspension prepared in the step S1 to make the concentration of the paraformaldehyde fixing solution be 0.4%, and inactivating the streptococcus agalactiae suspension at room temperature for 24 hours; washing thallus with PBS buffer solution for 4 times, centrifuging, mixing thallus precipitate with PBS buffer solution, and adjusting concentration of Streptococcus agalactiae to 1×10 8 The CFU/mL bacterial suspension is used for obtaining the inactivated streptococcus agalactiae antigen, and the streptococcus agalactiae antigen is put into a refrigerator at the temperature of 4 ℃ for standby;
s3, immunizing laying hens and collecting eggs; basic immunity and reinforcing immunity are carried out on the laying hen, eggs are collected every week after immunization, and the laying hen is stored in a refrigerator at 4 ℃ for standby;
s4, separating and purifying the egg yolk antibody; wiping the eggshell with alcohol, separating yolk with a yolk separator, adding precooled sterile distilled water with the yolk volume of 7 times into the yolk, adjusting the pH to 5.0 with 0.1M HCl, mixing, and standing overnight at 4deg.C in a refrigerator; centrifuging at 4deg.C and 12000g for 25min, collecting supernatant, and sequentially filtering with 0.45 μm and 0.2 μm microporous filter membrane to obtain water soluble component (WSF) of egg yolk antibody;
s5, purifying the egg yolk antibody; adding ammonium sulfate to the water-soluble component of the egg yolk antibody until the saturation is 55%, fully and uniformly stirring, centrifuging at 4 ℃ overnight under 12000g for 20min, diluting the precipitate with PBS buffer to the original volume, adding ammonium sulfate until the saturation is 33%, fully and uniformly stirring, standing at 4 ℃ for 2h, centrifuging at 4 ℃ under 12000g for 20min, and re-suspending the precipitate with PBS buffer to obtain the streptococcus agalactiae egg yolk antibody.
The application is further provided with: and (3) performing 1 basic immunization and 3 boosting immunization on the laying hens in the step (S3).
The application is further provided with: the basic immunization in the step S3 is to add equivalent Freund' S complete adjuvant into the streptococcus agalactiae antigen obtained in the step S1, fully emulsify and then perform pectoral muscle regional injection immunization on the laying hens, wherein the immunization dose is 1 mL/egg yolk immune.
The application is further provided with: and (3) enhancing immunity in the step (S3), adding equivalent Freund' S incomplete adjuvant into the streptococcus agalactiae antigen obtained in the step (S1), fully emulsifying, and carrying out pectoral muscle regional injection immunity on the laying hens, wherein the immunity dose is 1.5 mL/chicken, and the immunity interval is 10 days.
The application is further provided with: and after the anti-streptococcus agalactiae egg yolk antibody is obtained in the step S5, purity identification and activity measurement are carried out on the anti-streptococcus agalactiae egg yolk antibody, and the purity of the anti-streptococcus agalactiae egg yolk antibody is identified by adopting an enzyme-linked immunosorbent assay to detect the antibody titer and SDS-PAGE electrophoresis.
The application also provides an application of the streptococcus agalactiae egg yolk antibody, which comprises the step of preparing streptococcus agalactiae egg yolk antibody freeze-dried powder by using the streptococcus agalactiae egg yolk antibody; separating ovum gallus Domesticus flavus with ovum gallus Domesticus flavus separator, filtering ovum gallus Domesticus flavus solution with screen, pre-freezing at-20deg.C for more than 2 hr, and lyophilizing at-70deg.C under vacuum degree of 5Pa for 36 hr to obtain purified lyophilized powder of anti-Streptococcus agalactiae ovum gallus flavus antibody.
The application is further provided with: the streptococcus agalactiae-resistant egg yolk antibody or the streptococcus agalactiae-resistant egg yolk antibody freeze-dried powder is used for preparing medicines for infecting streptococcus agalactiae.
The application is further provided with: the anti-streptococcus agalactiae egg yolk antibody is used for preparing medicines for tilapia to infect streptococcus agalactiae.
In summary, the application has the following beneficial effects:
1. the anti-streptococcus agalactiae egg yolk antibody is obtained by immunizing an egg-laying hen with streptococcus agalactiae, has the advantages of good specificity, higher purity and high titer, has a stronger inhibiting effect on the streptococcus agalactiae, can be used for preventing and treating diseases caused by the streptococcus agalactiae, and can be used for detecting the streptococcus agalactiae immunology, and the titer detection method has high detection efficiency, accurate results and simple and convenient operation.
2. The streptococcus agalactiae-resistant egg yolk antibody adopts eggs produced by healthy hens as carriers, and has the advantages of simple preparation, safety, no toxicity, low cost, high yield and easy industrialization.
3. The streptococcus agalactiae-resistant egg yolk antibody prepared by the application has obvious protection effect and death delay effect on tilapia challenged by streptococcus agalactiae, and can be applied to preventing and treating diseases caused by streptococcus agalactiae of aquatic animals.
Drawings
Fig. 1: potency diagram of anti-streptococcus agalactiae egg yolk antibody.
Fig. 2: reduced SDS-PAGE gel electrophoresis of anti-streptococcus agalactiae egg yolk antibodies; wherein lane M: protein Maker; lane 1: a water soluble component of a yolk antibody; lane 2: the solution obtained after the first salting-out of ammonium sulfate; lane 3: and (5) salting out the solution obtained after the second time of ammonium sulfate salting out.
Fig. 3: western Blot images of egg yolk antibodies specific for Streptococcus agalactiae; wherein lane M: protein Maker; lane 1: an anti-streptococcus agalactiae egg yolk antibody; lane 2: a non-specific egg yolk antibody.
Fig. 4: scanning electron microscope images of the streptococcus agalactiae egg yolk antibody against streptococcus agalactiae; a and b are anti-streptococcus agalactiae egg yolk antibodies; c and d are non-specific egg yolk antibodies.
Fig. 5: confocal laser scanning microscopy images of streptococcus agalactiae by anti-streptococcus agalactiae egg yolk antibodies; a: an anti-streptococcus agalactiae egg yolk antibody; b: a non-specific egg yolk antibody; c: blank group.
Fig. 6: the egg yolk antibody for resisting streptococcus agalactiae has in-vitro antibacterial effect on streptococcus agalactiae.
Fig. 7: the anti-streptococcus agalactiae egg yolk antibody has therapeutic effect on tilapia infection streptococcus agalactiae.
Detailed Description
The application is described in further detail below with reference to fig. 1-7.
Examples: the application discloses preparation and application of a tilapia streptococcus agalactiae egg yolk antibody, which is shown in figures 1-7, wherein streptococcus agalactiae is taken as an antigen, an egg laying hen is immunized, and the egg yolk antibody for resisting streptococcus agalactiae is obtained by separating and extracting from immunized egg yolk; when the purity of the yolk antibody was detected by reducing SDS-PAGE, the pattern showed two bands, heavy and light chains of IgY, respectively. The embodiments are as follows:
1: preparation of streptococcus agalactiae antigen:
inoculating streptococcus agalactiae to a BHI solid agar culture medium, culturing for about 12 hours at 37 ℃, picking single colony, inoculating to a BHI liquid culture medium, shake culturing overnight at 37 ℃ and 160rpm, centrifuging to obtain a bacterial precipitate and supernatant containing streptococcus agalactiae, and washing the bacterial precipitate and supernatant with PBS buffer solution for 3 times to obtain the streptococcus agalactiae suspension.
2: inactivation of streptococcus agalactiae antigen:
to the suspension in step 1, 4% paraformaldehyde fixing solution was added to give a final concentration of 0.4% and the mixture was inactivated at room temperature for 24 hours. Washing thallus with PBS buffer solution for 4 times, centrifuging, mixing thallus precipitate with PBS buffer solution, and adjusting concentration of Streptococcus agalactiae to 1×10 8 The CFU/mL suspension is used for obtaining the inactivated streptococcus agalactiae antigen, and the streptococcus agalactiae antigen is put into a refrigerator at the temperature of 4 ℃ for standby.
3: immunizing laying hens and collecting eggs:
and (3) carrying out basic immunity and 3 times of boosting immunity on the laying hen, adding equivalent Freund's complete adjuvant into the inactivated streptococcus agalactiae antigen, and carrying out pectoral muscle regional injection immunization on the laying hen after full emulsification, wherein the immune dose is 1 mL/egg yolk antibody. And the boosting is to add equivalent Freund's incomplete adjuvant into the inactivated streptococcus agalactiae antigen, fully emulsify and then perform pectoral muscle regional injection immunization on the laying hens, wherein the immunization dose is 1.5 mL/egg yolk, and the immunization interval is 10 days. Eggs are collected every week after the first immunization, and are stored in a refrigerator at 4 ℃ for standby.
4: isolation of egg yolk antibodies from egg yolk
The immune egg is prepared by wiping eggshell with alcohol, separating yolk with yolk separator, adding pre-cooled sterile distilled water with yolk volume of 7 times, adjusting pH to 5.0 with 0.1M HCl, mixing, and standing overnight at 4deg.C. And centrifuging at 4deg.C and 12000g for 25min, and filtering the supernatant with 0.45 μm and 0.2 μm microporous filter membrane sequentially to obtain water soluble component (WSF) of egg yolk antibody.
5: purification of egg yolk antibody
Adding ammonium sulfate into the water-soluble component of the egg yolk antibody until the saturation is 55%, fully and uniformly stirring, centrifuging at 4 ℃ overnight and at 4 ℃ under 12000g for 20min, diluting the precipitate with PBS buffer to the original volume, adding ammonium sulfate until the saturation is 33%, fully and uniformly stirring, standing at 4 ℃ for 2h, centrifuging at 4 ℃ under 12000g for 20min, and re-suspending the precipitate with PBS buffer to obtain the anti-streptococcus agalactiae egg yolk antibody.
6: preparation of yolk antibody freeze-dried powder
Separating ovum gallus Domesticus flavus with ovum gallus Domesticus flavus separator, filtering ovum gallus Domesticus flavus solution with screen, pre-freezing at-20deg.C for more than 2 hr, and lyophilizing at-70deg.C under vacuum degree of 5Pa for 36 hr to obtain purified lyophilized powder of anti-Streptococcus agalactiae ovum gallus flavus antibody.
7: potency assay of egg yolk antibody against Streptococcus agalactiae
Formaldehyde-inactivated streptococcus agalactiae was adjusted to a concentration of 108CFU/mL with ELISA coating solution for coating of 96-well plates, 100 μl per well, and allowed to stand overnight in a refrigerator at 4 ℃. The next day the liquid in the wells was poured off, washed 3 times with PBST and then blocked with 200. Mu.L of blocking solution per well at 37℃for 2h. After 3 washes of PBST, WSF prepared in step 4 was diluted with antibody at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1: 25600. 1:51200 isocratic dilutions 100. Mu.L were added per well and incubated for 2h at 37℃to correspond toNon-specific WSF at fold dilutions was used as negative control, with 3 replicates per gradient. PBST is washed 3 times, 100 mu L of rabbit anti-chicken IgY marked by horseradish peroxidase with 1:100000 is added into each hole to be used as a secondary antibody, the secondary antibody is incubated for 1h at 37 ℃, TMB substrate color development liquid is added after washing 5 times, the secondary antibody is incubated for 20min at room temperature and in a dark place, and then 2mol/L H is added 2 SO 4 Stopping the reaction, reading the light absorption value with the wavelength of 450nm by using an enzyme-labeled instrument, wherein the ratio of the sample to be detected to the OD value of the negative control is more than or equal to 2.1, and the maximum dilution multiple of the corresponding sample is the titer of the anti-streptococcus agalactiae egg yolk antibody. As a result, as shown in FIG. 1, four red arrows represent four immunization times, each time being 10 days apart, and as shown in FIG. 1, the titer was slowly increased after the primary immunization, the titer was significantly increased after 4 weeks, the peak was reached (1:51200) after 6 weeks, and the period was continued for about 4 weeks, and then gradually decreased.
8: purity determination of egg yolk antibody against Streptococcus agalactiae
The products in the step 4 and the step 5 are taken for IgY purity identification, and detected by 10% reduction SDS-PAGE, and the result is shown in the attached figure 2, wherein, a lane M: protein Maker; lane 1: a water soluble component of a yolk antibody; lane 2: the solution obtained after the first salting-out of ammonium sulfate; lane 3: and (5) salting out the solution obtained after the second time of ammonium sulfate salting out. The results show that the yolk antibody consists of two heavy chains and two light chains, which are about 68kDa and about 25kDa respectively, and is consistent with literature reports.
9: western blot specificity detection of anti-streptococcus agalactiae egg yolk antibody
Streptococcus agalactiae was incubated with anti-Streptococcus agalactiae egg yolk antibodies and non-specific egg yolk antibodies for 2h at room temperature, boiled and centrifuged, and subjected to 10% reduction SDS-PAGE electrophoresis. The proteins on the gel were transferred to nitrocellulose membrane by semi-dry method, and the blocking solution (TBST solution containing 5% skimmed milk powder) was blocked for 2 hours at room temperature. Washed 5 times and then incubated with horseradish peroxidase-labeled rabbit anti-chicken IgY at 1:5000 dilution for 1h at room temperature. After 5 washes, color development was performed in the dark using ECL chemiluminescent hypersensitivity chromogenic reagent. The results are shown in figure 3, which shows that the secondary antibody can be specifically combined with the streptococcus agalactiae egg yolk antibody, and can be identified to display corresponding protein bands.
10: scanning electron microscope observation of influence of streptococcus agalactiae-resistant egg yolk antibody on streptococcus agalactiae surface structure
WSF of the anti-Streptococcus agalactiae egg yolk antibody and WSF of the non-specific egg yolk antibody were ultrafiltered with an ultrafiltration tube to a final concentration of 16mg/mL, mixed with BHI broth medium at equal ratio, and sterilized by filtration with a 0.2 μm microporous filter membrane. Regulating streptococcus agalactiae concentration to 10 6 CFU/mL, incubated at 37℃for 2h.4000g are centrifuged for 10min, washed 3 times by PBS, respectively added with 2mL of 2.5% glutaraldehyde fixing solution, fixed for 2h at room temperature, temporarily stored in a refrigerator at 4 ℃, and the samples are sent to the Wohai Weibull biotechnology Co. The results are shown in figure 4, which shows that streptococcus agalactiae bacteria under the action of the anti-streptococcus agalactiae egg yolk antibody have rough surfaces and irregular edges, and the bacteria are aggregated and adhered to each other.
11: confocal laser scanning microscope observation of affinity of egg yolk antibody against streptococcus agalactiae and streptococcus agalactiae
The concentration of the inactivated streptococcus agalactiae bacteria liquid is adjusted to be 10 6 CFU/mL was prepared by taking 750. Mu.L of bacterial solution, adding equal volumes of anti-Streptococcus agalactiae egg yolk antibody, non-specific egg yolk antibody (16 mg/mL) and PBS solution, and incubating at 37℃for 2h. Washed 3 times with PBS, 400. Mu.L of Fitc-labeled rabbit anti-chicken IgG (1:200 dilution) was added and incubated at 37℃for 2h in the absence of light. After 4 times of resuspension with PBS, 10. Mu.L of bacterial droplets were pipetted onto the slide and cover-slip fixed. The activity of the bacteria was examined with a confocal laser scanning microscope. As a result, in FIG. 5, more fluorescent labels were observed in the specific IgY group (FIG. 5 a) compared to the non-specific IgY group (FIG. 5 b) and the blank group (FIG. 5 c), indicating that specific IgY has a higher specific binding capacity with Streptococcus agalactiae and FITC-labeled IgG has a better affinity with IgY.
12: in vitro bacteriostasis test
The WSF of the anti-streptococcus agalactiae egg yolk antibody and the nonspecific egg yolk antibody are respectively concentrated by ultrafiltration with ultrafiltration concentration tubes, the antibody concentration is adjusted by an ultra-micro spectrophotometer to be 2mg/mL, 4mg/mL, 8mg/mL and 16mg/mL respectively, and a BHI broth culture medium without the egg yolk antibody is used as a blank control. All solutions were filter sterilized through a 0.2 μm microfiltration membrane. Taking Streptococcus agalactiae in logarithmic growth phase, and re-suspending with PBS to concentration of 10 7 CFU/mL, 3mL of bacterial liquid anddifferent concentrations of the anti-streptococcus agalactiae egg yolk antibody, the non-specific egg yolk antibody and the PBS solution are fully mixed at a ratio of 1:1. The mixture was subjected to shaking culture in a constant temperature shaker (160 rpm) at 37℃and 150. Mu.L of the mixture was taken every 2 hours to measure the absorbance at OD620 nm. The results are shown in figure 6, which shows that the prepared anti-streptococcus agalactiae egg yolk antibody has an inhibiting effect on streptococcus agalactiae and is dose-dependent.
13: application of anti-streptococcus agalactiae egg yolk antibody in treating tilapia infection streptococcus agalactiae
Healthy tilapia is selected and randomly divided into 4 groups of 21 tilapia groups, and the tilapia groups are intraperitoneally injected with 200 mu L of each group. Specific egg yolk antibody group: every milliliter (10) 7 CFU/mL) streptococcus agalactiae liquid is added with 10mg of streptococcus agalactiae yolk freeze-dried powder and mixed uniformly; non-specific egg yolk antibody group: every milliliter (10) 7 CFU/mL) streptococcus agalactiae liquid is added with 10mg of non-specific egg yolk antibody freeze-dried powder and mixed uniformly; positive control group: 10 7 CFU/mL; blank control group: no injection was made. And (5) conventional feeding, and observing survival conditions of 8d tilapia after toxicity attack. The results are shown in FIG. 7, and the survival rates of the non-specific egg yolk antibody group, the non-specific egg yolk antibody group and the positive control group after injection on day 1 were 81%, 28.6% and 19% respectively. The egg yolk antibody for resisting streptococcus agalactiae prepared by the application has obvious protection effect and death delay effect on tilapia challenged by streptococcus agalactiae, and can be applied to preventing and treating diseases caused by streptococcus agalactiae of aquatic animals.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (8)
1. A preparation method of an anti-streptococcus agalactiae egg yolk antibody is characterized by comprising the following steps: the method comprises the following steps:
s1, preparing streptococcus agalactiae antigen and inactivating; inoculating streptococcus agalactiae to a BHI solid agar culture medium, culturing for about 12 hours at 37 ℃ in an inversion way, selecting single bacterial colony, inoculating to the BHI liquid culture medium, shaking and culturing overnight at 37 ℃ and 160rpm, obtaining bacterial precipitate and supernatant containing streptococcus agalactiae through centrifugal separation, and washing bacterial precipitate for 3 times by using PBS buffer solution to obtain streptococcus agalactiae suspension;
s2: inactivation of streptococcus agalactiae antigen; adding 4% paraformaldehyde fixing solution into the streptococcus agalactiae suspension prepared in the step S1 to make the concentration of the paraformaldehyde fixing solution be 0.4%, and inactivating the streptococcus agalactiae suspension at room temperature for 24 hours; washing thallus with PBS buffer solution for 4 times, centrifuging, mixing thallus precipitate with PBS buffer solution, and adjusting concentration of Streptococcus agalactiae to 1×10 8 The CFU/mL bacterial suspension is used for obtaining the inactivated streptococcus agalactiae antigen, and the streptococcus agalactiae antigen is put into a refrigerator at the temperature of 4 ℃ for standby;
s3, immunizing laying hens and collecting eggs; basic immunity and reinforcing immunity are carried out on the laying hen, eggs are collected every week after immunization, and the laying hen is stored in a refrigerator at 4 ℃ for standby;
s4, separating and purifying the egg yolk antibody; wiping the eggshell with alcohol, separating yolk with a yolk separator, adding precooled sterile distilled water with the yolk volume of 7 times into the yolk, adjusting the pH to 5.0 with 0.1MHCl, mixing, and standing overnight at 4deg.C in a refrigerator; centrifuging at 4deg.C and 12000g for 25min, collecting supernatant, and sequentially filtering with 0.45 μm and 0.2 μm microporous filter membrane to obtain water soluble component (WSF) of egg yolk antibody;
s5, purifying the egg yolk antibody; adding ammonium sulfate to the water-soluble component of the egg yolk antibody until the saturation is 55%, fully and uniformly stirring, centrifuging at 4 ℃ overnight under 12000g for 20min, diluting the precipitate with PBS buffer to the original volume, adding ammonium sulfate until the saturation is 33%, fully and uniformly stirring, standing at 4 ℃ for 2h, centrifuging at 4 ℃ under 12000g for 20min, and re-suspending the precipitate with PBS buffer to obtain the streptococcus agalactiae egg yolk antibody.
2. The method for preparing the streptococcus agalactiae egg yolk antibody according to claim 1, which is characterized in that: and (3) performing 1 basic immunization and 3 boosting immunization on the laying hens in the step (S3).
3. The method for preparing the streptococcus agalactiae egg yolk antibody according to claim 1, which is characterized in that: the basic immunization in the step S3 is to add equivalent Freund' S complete adjuvant into the streptococcus agalactiae antigen obtained in the step S1, fully emulsify and then perform pectoral muscle regional injection immunization on the laying hens, wherein the immunization dose is 1 mL/egg yolk immune.
4. The method for preparing the streptococcus agalactiae egg yolk antibody according to claim 1, which is characterized in that: and (3) enhancing immunity in the step (S3), adding equivalent Freund' S incomplete adjuvant into the streptococcus agalactiae antigen obtained in the step (S1), fully emulsifying, and carrying out pectoral muscle regional injection immunity on the laying hens, wherein the immunity dose is 1.5 mL/chicken, and the immunity interval is 10 days.
5. The method for preparing the streptococcus agalactiae egg yolk antibody according to claim 1, which is characterized in that: and after the anti-streptococcus agalactiae egg yolk antibody is obtained in the step S5, purity identification and activity measurement are carried out on the anti-streptococcus agalactiae egg yolk antibody, and the purity of the anti-streptococcus agalactiae egg yolk antibody is identified by adopting an enzyme-linked immunosorbent assay to detect the antibody titer and SDS-PAGE electrophoresis.
6. The use of the anti-streptococcus agalactiae egg yolk antibody prepared by the preparation method according to claim 1, which is characterized in that: the streptococcus agalactiae egg yolk antibody is used for preparing streptococcus agalactiae egg yolk antibody freeze-dried powder; separating ovum gallus Domesticus flavus with ovum gallus Domesticus flavus separator, filtering ovum gallus Domesticus flavus solution with screen, pre-freezing at-20deg.C for more than 2 hr, and lyophilizing at-70deg.C under vacuum degree of 5Pa for 36 hr to obtain purified lyophilized powder of anti-Streptococcus agalactiae ovum gallus flavus antibody.
7. The use of the anti-streptococcus agalactiae egg yolk antibody of claim 6, wherein: the streptococcus agalactiae-resistant egg yolk antibody or the streptococcus agalactiae-resistant egg yolk antibody freeze-dried powder is used for preparing medicines for infecting streptococcus agalactiae.
8. The use of an anti-streptococcus agalactiae egg yolk antibody according to claim 7, characterized in that: the anti-streptococcus agalactiae egg yolk antibody is used for preparing medicines for tilapia to infect streptococcus agalactiae.
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