CN116751290A - Preparation method of vibrio vulnificus, vibrio fluvial and vibrio cholerae resisting yolk antibody - Google Patents
Preparation method of vibrio vulnificus, vibrio fluvial and vibrio cholerae resisting yolk antibody Download PDFInfo
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- CN116751290A CN116751290A CN202310756961.4A CN202310756961A CN116751290A CN 116751290 A CN116751290 A CN 116751290A CN 202310756961 A CN202310756961 A CN 202310756961A CN 116751290 A CN116751290 A CN 116751290A
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- vibrio
- yolk antibody
- vulnificus
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- cholerae
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1239—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Vibrionaceae (G)
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- 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
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- 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
-
- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/28—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Vibrionaceae (F)
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
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Abstract
The application belongs to the field of biotechnology, and discloses a preparation method of an anti-vibrio vulnificus, vibrio fluvialis and vibrio cholerae egg yolk antibody, which comprises the steps of S1, preparation and inactivation of antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae; s2, immunizing laying hens and collecting eggs; s3, separating and purifying the egg yolk antibody; and discloses a method for detecting the titer and purity of the yolk antibody, the growth inhibition effect of the yolk antibody on three vibrios, the affinity and agglutination effect of the yolk antibody on the three vibrios and the damage condition of the surface structure and the cell integrity of the yolk antibody on the three vibrios; also discloses the application of the yolk antibody in preparing medicines for preventing or treating diseases caused by three vibrios and in immunological detection; the yolk antibody prepared by the application has strong specificity and higher purity, has stronger inhibition effect on vibrio vulnificus, vibrio fluvialis and vibrio cholerae, has low preparation cost, is convenient and simple, and is suitable for large-scale production.
Description
Technical Field
The application relates to the technical field of biology, in particular to a preparation method of an anti-vibrio vulnificus, river vibrio and cholera vibrio yolk antibody.
Background
Vibrio vulnificus (Vibrio vulnificus), vibrio fluvialis (Vibrio fluvialis) and Vibrio cholerae (Vibrio cholerae) belong to Vibrionales (Vibrionales), vibrionaceae (Vibrionaceae), vibrio (Vibrio), are important pathogenic bacteria causing human and aquatic animal infections, and are widely existing in estuary, lake, seafood and coastal water environments around the world. Wherein, vibrio vulnificus is one of the most popular and serious pathogens of aquaculture bacterial diseases, mainly lodges in sea water, fish, shrimp, shellfish and plankton in warm sea areas, and is most suitable for propagation under the water temperature condition of 20-30 ℃; the fish infected by vibrio vulnificus mainly comprises tilapia, eel, grouper, cobia, spotted maigre, spearmint goby and the like, and can cause septicemia, putrescence and impetigo of various fishes, shrimps, crabs and shellfish, thus causing serious harm to the aquaculture industry; the vibrio cholerae can infect aquatic animals such as fishes, shrimps and the like, so that the aquatic animals are ill and die in a large amount, the fishes in the aquatic animals are regarded as natural hosts of the vibrio cholerae, the aquatic animals still have pathogenicity in a water environment with lower temperature, and the optimal growth temperature is 24-30 ℃; vibrio cholerae infects round fin tilapia, loach, anethole, white fish, and oncorhynchus mykiss, and bleeding symptoms, gill congestion, and eyeball rupture occur in the abdomen and fin base of diseased fish. In addition, after the vibrio cholerae infects carp and rainbow trout, effusion exists in the abdominal cavity of the fish body, and the bladder wall shows venous congestion symptoms and dies rapidly.
At present, the prevention and treatment means for diseases caused by vibrio vulnificus, vibrio fluvial and vibrio cholerae comprise: antibiotics and chemical drugs, chinese herbal medicines, inactivated vaccines, antibodies and the like. Among them, antibiotics and chemical drugs are widely used, however, with the long-term abuse of antibiotics and chemical drugs, the use of antibiotics and chemical drugs has the following problems:
1) Pathogenic bacteria generate drug resistance, so that the action effect of antibiotics is obviously reduced and even ineffective;
2) Serious water pollution is caused, and micro-ecological unbalance of the water environment is caused;
3) Antibiotic residues appear in the animal body, which affects the food quality safety of the aquatic products and indirectly endangers the health of human bodies.
The research shows that 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 can be used as 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 used as an effective method for preventing and controlling diseases. The method is characterized in that: compared with the existing antibiotics and chemical medicines, the application of the egg yolk antibody has the following advantages: the specificity is strong, and unlike antibiotics, the yolk antibody only aims at corresponding pathogenic bacteria and does not influence other flora; the acquisition is simple: the egg yolk antibody can be obtained from eggs of immunized laying hens, a large number of animals are not required to be killed, and the produced antibody has high titer and strong stability; a certain phylogenetic distance exists between the poultry and the mammal, and a small amount of antigen can trigger effective immune response; no residue: the egg yolk antibody belongs to dry immunoglobulin, and can be digested and absorbed by organisms as nutrient substances after the egg yolk antibody acts; short research and development period, low cost, long storage period and convenient transportation and application. Therefore, the egg yolk antibody is widely applied to the aspects of prevention and treatment of animal diseases, disease diagnosis and the like, and the U.S. 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 the like, and can also be applied to the fields of immunological detection, aquatic product preservation, disease diagnosis and the like.
However, the prior egg yolk antibody is mainly applied to the field virus of the macrobrachium rosenbergii, the cryptosporidiosis of the behcet, the vibrio fludrosophila, the aeromonas hydrophila, the vibrio cholerae, the vibrio lautus and the vibrio anguillarum and the like, and the egg yolk antibody applied to the three simultaneously has not been reported.
Disclosure of Invention
The application aims to provide a preparation method of the vibrio vulnificus, fluvial vibrio and cholera resisting egg yolk antibody, the prepared triple egg yolk antibody has strong specificity and higher purity, and has stronger inhibition effect on vibrio vulnificus, fluvial vibrio and cholera, and the preparation cost of the triple egg yolk antibody is low, convenient and simple, and is suitable for large-scale production. The problems are effectively solved.
In order to achieve the above object, the present application provides the following technical solutions:
the preparation method of the vibrio vulnificus, vibrio fluvial and vibrio cholerae resisting yolk antibody comprises the following steps:
s1, preparation and inactivation of antigens of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae
Inoculating vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a liquid culture medium respectively, then placing the vibrio vulnificus, the vibrio fluvialis and the vibrio cholerae in a constant-temperature shaking table respectively for shaking culture overnight, centrifugally collecting thalli, and washing the collected thalli by using a buffer solution to obtain a bacterial suspension;
respectively adding a fixing solution into the bacterial suspension to adjust the concentration of the bacterial suspension, inactivating the bacterial suspension at room temperature, washing and centrifuging the bacterial suspension, uniformly mixing bacterial precipitate obtained by centrifugation with a buffer solution, finally mixing vibrio vulnificus, vibrio fluvialis and vibrio cholerae, adjusting the concentration of the bacterial suspension to obtain inactivated antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae, and refrigerating the bacterial suspension for later use;
s2, 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 eggs are refrigerated for standby;
s3, separation and purification of yolk antibody
S3.1, sterilizing eggshells of the immunized eggs obtained in the step S2, separating yolk, adding precooled sterile distilled water into the yolk, adjusting pH, stirring the mixed solution uniformly, and standing overnight;
s3.2, centrifuging the mixed solution obtained in the step S3.1, and filtering the obtained supernatant by using a microporous filter membrane to obtain a water-soluble component of the egg yolk antibody;
s3.3, regulating the saturation of the water-soluble component of the egg yolk antibody in the step S3.2, and standing overnight after the mixed solution is stirred uniformly;
s3.4, centrifuging the mixed solution obtained in the step S3.3, diluting the obtained precipitate to the original volume by using a buffer solution, adjusting the saturation again, stirring the mixed solution uniformly, and standing for 2 hours;
s3.5, centrifuging the mixed solution obtained in the step S3.4, and re-suspending the precipitate by using a buffer solution to obtain the triple egg yolk antibody.
Further, in S1, the liquid medium used for inoculation is 2216E liquid medium; the temperature of the constant temperature shaking table is 30 ℃; the fixing solution is 4% paraformaldehyde fixing solution; the final concentration of the bacterial suspension is 0.4%, and the room temperature inactivation time is 24 hours; the buffer solution for flushing the thalli is BS buffer solution; the volume ratio of mixing Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae is 1:1:1, a step of; the concentration of the bacterial suspension is 3x108CFU/mL; the refrigerating temperature was 4 ℃.
Further, in S2, the number of times of basic immunization is 1, and the method of basic immunization is as follows: adding equivalent Freund' S complete adjuvant into the antigens of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae obtained in the step S1, and performing pectoral region injection immunization on the laying hen after full emulsification, wherein the immunization dose is 1 mL/egg chicken;
the number of times of boosting is 3, and the method for boosting is as follows: adding the antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae obtained in the step S1 into an equivalent amount of incomplete adjuvant, fully emulsifying, and carrying out pectoral region injection immunization on the laying hens, wherein the immunization dosage is 1.5 mL/chicken, and the immunization interval is 10 days;
the temperature of the cold storage of the eggs is 4 ℃.
Further, in S3.1, the eggshells are wiped with alcohol to be sterilized, and the yolk is separated by a yolk separator; adding precooled sterile distilled water with the volume being 7 times of the yolk volume; the pH concentration of the mixed solution is adjusted to 5.0 by using 0.1M HCl, and the temperature of standing overnight is 4 ℃;
in S3.2, the mixed solution is centrifuged at 12000g for 25min at 4 ℃, and the pore diameter of the microporous filter membrane is 0.45 μm and 0.2 μm;
in S3.3, regulating the saturation of the mixed solution to 55% by using ammonium sulfate, and standing overnight at 4 ℃;
in S3.4, centrifuging the mixed solution at 4 ℃ for 20min under 12000g, diluting the precipitate by using PBS buffer solution, regulating the saturation of the mixed solution to 33% by using ammonium sulfate, and standing at 4 ℃;
in S3.5, the mixed solution was centrifuged at 12000g for 20min at 4℃and the pellet was resuspended in PBS buffer.
The triple egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae is prepared by the preparation method of the egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae.
The method for detecting the triple egg yolk antibody against vibrio vulnificus, vibrio fluvialis and vibrio cholerae, which is prepared by the preparation method of the egg yolk antibody against vibrio vulnificus, vibrio fluvialis and vibrio cholerae, adopts an enzyme-linked immunosorbent assay to detect the antibody titer and SDS-PAGE electrophoresis to identify the purity of the antibody; the growth inhibition effect of the triple yolk antibody on vibrio vulnificus, vibrio fluvialis and vibrio cholerae is observed through an in vitro bacteriostasis test; observing the affinity and agglutination effects of the triple yolk antibody on vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a confocal laser scanning microscope; and observing the damage condition of the triple yolk antibody to the surface structure and the cell integrity of vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a scanning electron microscope.
The triple egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae, which is prepared by the preparation method of the egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae, is applied to the preparation of medicaments for preventing or treating diseases caused by vibrio vulnificus, vibrio fluvialis and vibrio cholerae.
The triple egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae, which is prepared by the preparation method of the egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae, is applied to immunological detection of vibrio vulnificus, vibrio fluvialis and vibrio cholerae.
The technical proposal has the beneficial effects that:
1. the triple egg yolk antibody for resisting vibrio vulnificus, vibrio fluvialis and vibrio cholerae, which is prepared by immunizing healthy egg laying hens with antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae, has the advantages of good specificity, higher purity and high titer;
2. the prepared triple egg yolk antibody has a strong inhibition effect on vibrio vulnificus, vibrio fluvialis and vibrio cholerae, and can be used for preventing and treating diseases caused by vibrio vulnificus, vibrio fluvialis and vibrio cholerae and detecting immunology;
3. the method for preparing the triple egg yolk antibody against vibrio vulnificus, vibrio fluvialis and vibrio cholerae is simple, safe and nontoxic, low in cost, high in yield and easy to industrialize.
Drawings
FIG. 1 is a potency chart of a triple egg yolk antibody prepared in accordance with an embodiment of the present application;
in the figure, A is vibrio vulnificus; b is Vibrio fluvialis; c is Vibrio cholerae
FIG. 2 is a diagram showing the reduced SDS-PAGE gel of the triple egg yolk antibodies prepared in the examples of the present application;
in the figure, M is a protein Maker;1 is WSF;2 is first salting out; 3 is salting out for the second time
FIG. 3 is a Western Blot diagram of a triple yolk antibody prepared according to an embodiment of the present application;
in the figure, A is vibrio vulnificus; b is Vibrio fluvialis; c is Vibrio cholerae; m is a protein Maker;1 is a triple egg yolk antibody; 2 is non-specific egg yolk antibody
FIG. 4 is a scanning electron microscope image of the triple yolk antibody prepared by the embodiment of the application on Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae;
in the figure, A is vibrio vulnificus; b is Vibrio fluvialis; c is Vibrio cholerae; (a) is a triple egg yolk antibody; (b) Is a non-specific egg yolk antibody
FIG. 5 is a confocal laser scanning microscope image of the triple egg yolk antibody prepared by the embodiment of the application on Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae;
in the figure, A is vibrio vulnificus; b is Vibrio fluvialis; c is Vibrio cholerae; (a) is a triple egg yolk antibody; (b) is a non-specific egg yolk antibody; (c) Is blank group
FIG. 6 is a graph showing the in vitro bacteriostatic effect of the triple yolk antibody prepared in the embodiment of the application on Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae;
in the figure, A is vibrio vulnificus; b is Vibrio fluvialis; c is Vibrio cholerae
Detailed Description
The application is described in further detail below with reference to the attached drawings and embodiments:
the preparation method of the vibrio vulnificus, vibrio fluvial and vibrio cholerae resisting yolk antibody comprises the following steps:
preparation of Vibrio antigen
Inoculating vibrio vulnificus, vibrio fluvialis and vibrio cholerae into 2216E liquid culture medium, shake culturing at 30deg.C with shaking table overnight, centrifuging to collect thallus, and washing thallus with PBS buffer solution for 3 times to obtain vibrio suspension;
inactivation of Vibrio antigen
And respectively adding 4% paraformaldehyde fixed solution into the bacterial suspension to make the final concentration of the paraformaldehyde fixed solution be 0.4%, and inactivating the bacterial suspension at room temperature for 24 hours. Washing thallus with PBS buffer solution for 4 times, mixing the centrifuged thallus precipitate with PBS buffer solution, mixing Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae at a ratio of 1:1, and adjusting the concentration of the thallus to 3x10 8 CFU/mL to obtain inactivated antigens of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae, and placing in a refrigerator at 4 ℃ for later use;
immunizing laying hen and collecting eggs
1-time basic immunization and 3-time boosting immunization are carried out on the laying hen, equivalent Freund's complete adjuvant is added into the inactivated vibrio antigen, after full emulsification, the breast muscle is injected and immunized on the laying hen, and the immunization dose is 1 mL/egg yolk; the boosting is to add equivalent Freund's incomplete adjuvant into the inactivated vibrio antigen, fully emulsify and then to perform chest muscle regional injection immunization on the laying hen, wherein the immunization dose is 1.5 mL/chicken, and the immunization interval is 10 days. Collecting eggs every week after the first immunization, and storing in a refrigerator at 4 ℃ for later use;
isolation of egg yolk antibodies from egg yolk
Wiping eggshells of immunized eggs with alcohol, separating yolk by a yolk separator, adding precooled sterile distilled water with the yolk volume of 7 times, adjusting pH to 5.0 with 0.1M HCl, mixing, and standing at 4deg.C overnight. 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.
Purification of 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 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 ℃ and at 12000g for 20min, and re-suspending the precipitate with PBS buffer to obtain the triple egg yolk antibody.
Example 1
Titer determination of triple yolk antibodies against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae
Formaldehyde inactivated Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae are respectively adjusted to 10 in concentration by ELISA coating liquid 8 CFU/mL, used for coating of 96-well plates, 100. Mu.L per well, and left standing 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, antibody dilutions for WSF prepared above were used at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1: 25600. 1:51200 equi-gradient dilutions were added to each well and incubated at 37℃for 2h, with a negative control of nonspecific WSF at corresponding dilution, 3 replicates per gradient. PBST washing 3 times100 mu L of rabbit anti-chicken IgY marked by horseradish peroxidase with the concentration of 1:100000 is added into each hole to be used as a secondary antibody, incubated for 1h at 37 ℃, TMB substrate color development liquid is added after washing for 5 times, 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-vibrio fluvialis egg yolk antibody.
As shown in fig. 1, 4 arrows represent the time of primary immunization, secondary immunization, tertiary immunization, and quaternary immunization, respectively, each time being 10 days apart; as can be seen from FIG. 1, the titer was slowly increased after the primary immunization, the titer was significantly increased after 4 weeks, the peak was reached after 6 weeks (1:51200), the duration was about 4 to 5 weeks, and then gradually decreased.
Example two
Purity determination of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae triple yolk antibody
The water-soluble component of the egg yolk antibody and the triple egg yolk antibody prepared by the method are taken for IgY purity identification, and 15% reduction SDS-PAGE detection is used.
As shown in fig. 2, lane M is a protein Marker; lane 1 is the water soluble fraction of egg yolk antibody (WSF); lane 2 is the solution obtained after the first salting-out of ammonium sulfate; lane 3 shows the solution obtained after a second salting-out of ammonium sulfate. As can be seen from fig. 2, the egg yolk antibody consists of two heavy and light chains of about 68kDa and about 25kDa, respectively.
Example III
Westernblot specificity detection of triple egg yolk antibodies against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae
Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae are respectively incubated with the triple egg yolk antibody and the nonspecific egg yolk antibody for 2 hours at room temperature, boiled and centrifuged, and 10% reduced SDS-PAGE is performed. 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.
As shown in FIG. 3, the secondary antibody can be specifically combined with the triple yolk antibody, and can be recognized to display corresponding protein bands.
Example IV
Influence of triple yolk antibody on surface structures of vibrio vulnificus, fluvial vibrio and cholera vibrio observed by scanning electron microscope
The WSF of the triple egg yolk antibody and the WSF of the nonspecific egg yolk antibody were ultrafiltered with an ultrafiltration tube to a final concentration of 16mg/mL, mixed with 2216E liquid medium at equal ratio, and sterilized by filtration with a 0.2 μm microporous filter membrane. Respectively adjusting the concentration of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae 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.
As shown in FIG. 4, the cell surfaces of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae under the action of the triple yolk antibody were rough, the edges were irregular, and the cells were aggregated and adhered to each other, as compared with the non-specific yolk antibody.
Example five
Confocal laser scanning microscope for observing affinity of triple yolk antibody to Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae
The concentration of the bacterial liquid is respectively adjusted to 10 by inactivated vibrio vulnificus, vibrio fluvial and vibrio cholerae 6 CFU/mL was prepared by taking 750. Mu.L of bacterial liquid, adding equal volumes of triple egg yolk antibody, nonspecific egg yolk antibody (16 mg/mL) and PBS solution, and incubating at 37℃for 2h. Washed 3 times with PBS, and incubated with 400. Mu. LFItc labeled rabbit anti-chicken IgG (1:200 dilution) 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 shown in fig. 5, more fluorescent markers were observed in the prepared triple egg yolk antibody group (fig. 5 a) compared with the non-specific egg yolk antibody group (fig. 5 b) and the blank group (fig. 5 c), indicating that the prepared triple egg yolk antibody has stronger specific binding ability with vibrio vulnificus, vibrio fluvialis and vibrio cholerae, and FITC-labeled IgG has better affinity with IgY.
Example six
In vitro bacteriostasis test
The WSF of the triple egg yolk antibody and the WSF of the nonspecific egg yolk antibody are respectively concentrated by ultrafiltration with an ultrafiltration concentration tube, the antibody concentration is adjusted by an ultra-micro spectrophotometer to be 2mg/mL, 4mg/mL, 8mg/mL and 16mg/mL respectively, and 2216E liquid 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. Respectively taking Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae in logarithmic growth phase, and re-suspending with PBS to concentration of 10 5 CFU/mL, 3mL of bacterial liquid is taken and fully mixed with triple egg yolk antibody, nonspecific egg yolk antibody and PBS solution with different concentrations at a ratio of 1:1. Shaking culture at 30deg.C with a constant temperature shaker (160 rpm), taking 150 μl every 2h, and measuring OD620 nm Absorbance.
As shown in FIG. 6, it was revealed that the prepared triple egg yolk antibody had a remarkable inhibitory effect on Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae, and exhibited dose dependence.
The foregoing is merely exemplary embodiments of the present application, and detailed technical solutions or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, and these should also be regarded as the protection scope of the present application, which does not affect the effect of the implementation of the present application and the practical applicability of the patent. The protection scope of the present application is subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (8)
1. The preparation method of the vibrio vulnificus, vibrio fluvial and vibrio cholerae resisting yolk antibody is characterized by comprising the following steps of:
s1, preparation and inactivation of antigens of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae
Inoculating vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a liquid culture medium respectively, then placing the vibrio vulnificus, the vibrio fluvialis and the vibrio cholerae in a constant-temperature shaking table respectively for shaking culture overnight, centrifugally collecting thalli, and washing the collected thalli by using a buffer solution to obtain a bacterial suspension;
respectively adding a fixing solution into the bacterial suspension to adjust the concentration of the bacterial suspension, inactivating the bacterial suspension at room temperature, washing and centrifuging the bacterial suspension, uniformly mixing bacterial precipitate obtained by centrifugation with a buffer solution, finally mixing vibrio vulnificus, vibrio fluvialis and vibrio cholerae, adjusting the concentration of the bacterial suspension to obtain inactivated antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae, and refrigerating the bacterial suspension for later use;
s2, 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 eggs are refrigerated for standby;
s3, separation and purification of yolk antibody
S3.1, sterilizing eggshells of the immunized eggs obtained in the step S2, separating yolk, adding precooled sterile distilled water into the yolk, adjusting pH, stirring the mixed solution uniformly, and standing overnight;
s3.2, centrifuging the mixed solution obtained in the step S3.1, and filtering the obtained supernatant by using a microporous filter membrane to obtain a water-soluble component of the egg yolk antibody;
s3.3, regulating the saturation of the water-soluble component of the egg yolk antibody in the step S3.2, and standing overnight after the mixed solution is stirred uniformly;
s3.4, centrifuging the mixed solution obtained in the step S3.3, diluting the obtained precipitate to the original volume by using a buffer solution, adjusting the saturation again, stirring the mixed solution uniformly, and standing for 2 hours;
s3.5, centrifuging the mixed solution obtained in the step S3.4, and re-suspending the precipitate by using a buffer solution to obtain the triple egg yolk antibody.
2. The method for preparing an anti-vibrio vulnificus, vibrio fluvial and vibrio cholerae egg yolk antibody according to claim 1, wherein in S1, the liquid medium for inoculation is 2216E liquid medium; the temperature of the constant temperature shaking table is 30 ℃; the fixing solution is 4% paraformaldehyde fixing solution; the final concentration of the bacterial suspension is 0.4%, and the room temperature inactivation time is 24 hours; the buffer solution for flushing the thalli is BS buffer solution; the volume ratio of mixing Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae is 1:1:1, a step of; the concentration of the bacterial suspension is 3x108CFU/mL; the refrigerating temperature was 4 ℃.
3. The method for preparing the yolk antibody against vibrio vulnificus, vibrio fluvial and vibrio cholerae according to claim 1, wherein in S2, the number of basic immunizations is 1, and the method for basic immunizations is as follows: adding equivalent Freund' S complete adjuvant into the antigens of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae obtained in the step S1, and performing pectoral region injection immunization on the laying hen after full emulsification, wherein the immunization dose is 1 mL/egg chicken;
the number of times of boosting is 3, and the method for boosting is as follows: adding the antigens of vibrio vulnificus, vibrio fluvialis and vibrio cholerae obtained in the step S1 into an equivalent amount of incomplete adjuvant, fully emulsifying, and carrying out pectoral region injection immunization on the laying hens, wherein the immunization dosage is 1.5 mL/chicken, and the immunization interval is 10 days;
the temperature of the cold storage of the eggs is 4 ℃.
4. The method for preparing an anti-vibrio vulnificus, vibrio fluvialis and vibrio cholerae yolk antibody according to claim 1, wherein in S3.1, the eggshells are wiped with alcohol for sterilization, and the yolk is separated by a yolk separator; adding precooled sterile distilled water with the volume being 7 times of the yolk volume; adjusting the pH concentration of the mixed solution to 5.0 by using 0.1MHCl, and standing overnight at 4 ℃;
in S3.2, the mixed solution is centrifuged at 12000g for 25min at 4 ℃, and the pore diameter of the microporous filter membrane is 0.45 μm and 0.2 μm;
in S3.3, regulating the saturation of the mixed solution to 55% by using ammonium sulfate, and standing overnight at 4 ℃;
in S3.4, centrifuging the mixed solution at 4 ℃ for 20min under 12000g, diluting the precipitate by using PBS buffer solution, regulating the saturation of the mixed solution to 33% by using ammonium sulfate, and standing at 4 ℃;
in S3.5, the mixed solution was centrifuged at 12000g for 20min at 4℃and the pellet was resuspended in PBS buffer.
5. The triple egg yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae prepared by the method of preparing the egg yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae according to any one of claims 1 to 4.
6. The method for detecting the triple egg yolk antibodies against vibrio vulnificus, vibrio fluvialis and vibrio cholerae prepared by the method for preparing the egg yolk antibodies against vibrio vulnificus, vibrio fluvialis and vibrio cholerae according to any one of claims 1 to 4, wherein the method is characterized in that the antibody titer and SDS-PAGE electrophoresis are detected by an enzyme-linked immunosorbent assay to identify the purity of the antibodies; the growth inhibition effect of the triple yolk antibody on vibrio vulnificus, vibrio fluvialis and vibrio cholerae is observed through an in vitro bacteriostasis test; observing the affinity and agglutination effects of the triple yolk antibody on vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a confocal laser scanning microscope; and observing the damage condition of the triple yolk antibody to the surface structure and the cell integrity of vibrio vulnificus, vibrio fluvialis and vibrio cholerae by using a scanning electron microscope.
7. The use of a triple egg yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae prepared by the method for preparing a yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating diseases caused by Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae.
8. The use of a triple yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae prepared by the method for preparing a yolk antibody against Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae according to any one of claims 1 to 4 in immunological detection of Vibrio vulnificus, vibrio fluvialis and Vibrio cholerae.
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