CN112175076A - Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection - Google Patents
Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection Download PDFInfo
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- CN112175076A CN112175076A CN202010725674.3A CN202010725674A CN112175076A CN 112175076 A CN112175076 A CN 112175076A CN 202010725674 A CN202010725674 A CN 202010725674A CN 112175076 A CN112175076 A CN 112175076A
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- vibrio vulnificus
- yolk antibody
- yolk
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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
- C07K16/1203—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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- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
Abstract
The invention provides a yolk antibody for preventing and treating vibrio vulnificus infection, which is prepared by the following method: (1) isolating Vibrio vulnificus from a sample of a Vibrio vulnificus infected patient in a natural environment; (2) preparing a vibrio vulnificus inactivated bacterial antigen and immunizing an egg-laying hen; (3) collecting the immunized eggs, and extracting and purifying yolk antibodies from the yolk of the eggs. The yolk antibody of the invention can effectively neutralize vibrio vulnificus so as to stop vibrio vulnificus infection, and provides an effective passive immune preparation for subsequent prevention and control of the bacteria.
Description
Technical Field
The invention belongs to the technical field of biological product production, and particularly relates to a preparation method of a yolk antibody for preventing and treating vibrio vulnificus infection.
Background
Along with the continuous progress of social economy, the human needs for various resources are more and more. The ocean contains extremely rich resources, and people are promoted to gradually project eyes to the ocean. With the emphasis on ocean resources and shipping in various countries, the ocean economic activities of human beings are increasingly frequent. The incidence of marine-related infectious diseases has also increased year by year.
Vibrio vulnificus is a lethal marine vibrio. The amputation rate and the fatality rate are high. At present, the treatment means comprises traditional wound debridement and the use of antibiotics, and the death risk cannot be effectively reduced. The vibrio has the highest detection rate in marine bacteria detection and is the dominant flora in the sea. Among them, only a few Vibrio species infect humans, and Vibrio vulnificus infection is the most fatal. Vibrio vulnificus is a gram-negative opportunistic pathogen of halophilic nature. It is widely distributed in the water areas of the river and sea in the world, and is most common in low-latitude areas with high water temperature. It can invade human body through oral or damaged skin, thus causing symptoms such as human body primary septicemia, gastrointestinal tract septicemia and local wound infection. The vibrio vulnificus infected condition is dangerous and the disease course is rapidly progressed.
The main treatment means at present comprise traditional debridement and the use of antibiotics, and although vibrio vulnificus is sensitive to most antibiotics, early diagnosis and timely use of antibiotics cannot be realized due to negligence of patients or limited injured environment. In addition to traditional debridement, topical wounds lack other effective treatment means, and topical use of antibiotics has been shown to be susceptible to flora resistance, especially in patients with gastrointestinal infections, where the local treatment means is more limited due to the presence of lesions in the body.
In clinical treatment strategies, early diagnosis and early treatment are of great significance to the prognosis of Vibrio vulnificus infection. Unfortunately, although Vibrio vulnificus is sensitive to most antibiotics, it is subject to various factors such as patient negligence or limited environment of injury. Vibrio vulnificus infection is often not diagnosed early and treated with antibiotics and the like in time. The wide-range preventive use of antibiotics will cause the effect of Vibrio vulnificus drug resistance. At present, due to the abuse of antibiotics in aquaculture industry worldwide, more and more countries and regions report the problem of drug resistance of vibrio vulnificus. In addition, topical wound treatment with antibiotics has proven to be easily abandoned in the cause of flora resistance. In addition to traditional debridement, topical wounds lack more therapeutic approaches. For patients with gastrointestinal infections, local treatment is more limited because the lesions are located in the abdominal cavity. Clinical treatment means capable of avoiding selective drug resistance of strains is urgently needed.
Passive immunization is an important component of immunotherapy. Passive immunization, such as antibody therapy, is increasingly gaining attention today due to its ability to circumvent selective resistance in cases of severe antibiotic abuse. The process for extracting and preparing the specific IgG from the serum of the mammal is complex, the cost is high, and the large-scale industrialized production and application are difficult to realize.
Disclosure of Invention
The yolk antibody IgY is a corresponding specific antibody produced by poultry stimulated by environment specific antigen. IgY in laying fowl can be enriched into yolk by circulation to protect embryo in egg from attack by pathogenic microorganisms in the environment. The yolk antibody has different action principles with antibiotic drugs, and mainly combines with an antigen or hapten site carried by pathogenic microorganisms, so that the original functions of the antigen site are changed or prevented, and the biological activity of the pathogenic microorganisms is further influenced, such as inhibiting the normal division and propagation of the pathogenic microorganisms, influencing the adhesion of the pathogenic microorganisms to normal tissues, destroying the formation of a biological membrane or inactivating the secreted virulence factors and the like. Eventually, the antigen-antibody complex will be cleared by the body's immune system. The egg yolk antibody IgY has been widely used in aquaculture and animal husbandry, but has not been used for preventing and treating human diseases. The invention firstly proposes that the chicken egg yolk antibody IgY is used for preventing and treating human infectious diseases.
The invention aims to provide the chicken egg yolk antibody which has quick response, economy, high efficiency and no obvious toxic or side effect, and can be widely used for preventing and treating vibrio vulnificus infection without causing selective drug resistance of strains. Another purpose is to provide a preparation method of the chicken egg yolk antibody against Vibrio vulnificus.
In order to achieve the purpose, the invention adopts the technical scheme that: a yolk antibody for preventing and treating Vibrio vulnificus infection is prepared by the following method, comprising the following steps:
(1) isolating Vibrio vulnificus from a sample of a Vibrio vulnificus-infected patient;
(2) preparing a vibrio vulnificus inactivated bacterial antigen and immunizing an egg-laying hen;
(3) collecting the immunized eggs, and extracting and purifying yolk antibodies from the yolk of the eggs.
Further, the step (1) of isolating Vibrio vulnificus from the sample of the Vibrio vulnificus-infected patient includes the specific steps of: extracting and identifying vibrio vulnificus strain, separating and purifying, culturing and storing.
Further, the specific operations of the extraction, identification, separation, purification, culture and preservation of the vibrio vulnificus strain are as follows: selecting wound surface blister liquid or tissue exudate of a patient infected with vibrio vulnificus clinically, diluting the wound surface blister liquid or tissue exudate, coating a plate, and culturing overnight; selecting a single colony, determining whether the colony is infected by the vibrio vulnificus by a mass spectrum analyzer, and performing amplification culture on the strain determined to be the vibrio vulnificus.
Further, the plates were blood plates to which 0.5% sodium chloride was added.
Further, the temperature of the culture overnight was 37 ℃.
Further, the step (2) of preparing the inactivated bacteria antigen of vibrio vulnificus comprises the following specific steps: inactivating the vibrio vulnificus by using a formaldehyde inactivation method to prepare a fully inactivated thallus antigen.
The formaldehyde inactivation method specifically comprises the following steps: the inactivating concentration of formaldehyde is 0.1-0.3%, the inactivating time is 24 hours, and the inactivating temperature is 35-37 ℃.
Further, the specific steps of immunizing the laying hens in the step (2) are as follows: the antigen solution is taken to carry out intramuscular injection on the deltoid muscle or the pectoralis muscle of the wing base part of the hen, and the inoculation is carried out once every two weeks.
Further, the step (2) also comprises the step of detecting the titer of the yolk antibodies generated after the egg laying hens are immunized.
Further, the specific operation of extracting and purifying the yolk antibody from the yolk of the egg in the step (3) is as follows:
(1) mixing and dissolving yolk water into homogenate, adjusting the pH value of the homogenate to 5.0 +/-0.5, and cooling the acidified homogenate solution to 2-8 ℃ and standing for 1-12 hours;
(2) keeping the temperature of 2-8 ℃ and centrifuging the homogenate at a high speed of 5000-;
(3) adding ammonium sulfate into the supernatant, centrifuging, and collecting precipitate to obtain crude extract;
(4) and (3) redissolving the crude extract in a weak acid buffer solution, and concentrating the redissolved solution through ultrafiltration membranes of 300kD and 100kD in sequence to obtain a yolk antibody mixed concentrated solution.
Further, the mass concentration of the ammonium sulfate is 15-45%.
Further, the step of extracting and purifying the yolk antibody from the yolk of the egg in the step (3) further comprises detecting the titer of the yolk antibody and adding a protein stabilizer.
The invention also provides application of the yolk antibody of the inactivated thallus of vibrio vulnificus in preparing a reagent or a medicament for preventing or/and treating vibrio vulnificus infection.
The invention has the beneficial effects that: the inactivated thallus antibody of vibrio vulnificus has great amount of specific antibody to vibrio conservative protein, so that it has binding effect on all vibrio vulnificus individuals and no selective effect exists theoretically. Under the severe situation that multiple drug resistance of pathogenic microorganisms is caused by the current abuse of antibiotics, the application range of the non-selective inhibition specific antibody passive immunotherapy is wider.
Detailed Description
In order to more concisely and clearly demonstrate technical solutions, objects and advantages of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example (b):
a yolk antibody for preventing and treating Vibrio vulnificus infection is prepared by the following method:
1. extracting, identifying, separating, purifying, culturing and storing the clinical highly pathogenic vibrio vulnificus strain:
selecting patients with vibrio vulnificus infection clinically, and taking wound surface blister liquid or tissue exudate. Plates were plated on blood plates to which 0.5% sodium chloride was added. The cells were placed in a 37 ℃ incubator overnight. After single colony is picked and centrifugally purified, a mass spectrum analyzer is used for determining whether the infection is caused by the vibrio vulnificus. After diagnosis, the strain is cultured and amplified and stored for later use.
2. Preparing a vibrio vulnificus inactivated thallus antigen:
inactivating the vibrio vulnificus by using a formaldehyde inactivation method to prepare a fully inactivated thallus antigen. Performing multiple immunization and yolk antibody titer detection on the laying hens;
selecting healthy laying hens of 12 weeks old, and injecting antigen solution at multiple sites of deltoid muscle or pectoral muscle of chicken at multiple points such as muscle and subcutaneous tissue. The inoculation is carried out once every two weeks, and sampling is carried out every two weeks to detect the antibody titer in the egg yolk. Until titer is exceeded-vaccination is stopped. Antibody titers in the yolk were continued every two weeks.
3. Collecting and sterilizing immune eggs, and separating, extracting and concentrating yolk antibodies;
collecting the eggs of the standard-reaching batch, flushing the eggs with flowing water, and spraying 75% alcohol on the surfaces of the eggs for disinfection. The yolk is separated and obtained by using a sterile egg separator. Mixing yolk with purified water at a ratio of 1:5-10, dissolving to obtain homogenate, and adjusting pH value of the homogenate to 5.0 + -0.5. Cooling the acidified homogenate solution to 2-8 deg.C, and standing for 1-12 hr. Then keeping the low temperature environment to centrifuge the homogenate at the high speed of 5000-. Removing supernatant, and discarding the rest liquid. Adding 15% -45% ammonium sulfate into the obtained supernatant for salting out, and centrifuging to obtain precipitate crude extract. The crude extract was then redissolved in a weakly acidic buffer (0.2% glycine). Finally, the redissolution is concentrated by sequentially passing through 300kD and 100kD ultrafiltration membranes. And (4) obtaining a yolk antibody mixed concentrated solution.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A yolk antibody for preventing and treating Vibrio vulnificus infection, which is prepared by the following method:
(1) isolating Vibrio vulnificus from a sample of a Vibrio vulnificus-infected patient;
(2) preparing a vibrio vulnificus inactivated bacterial antigen and immunizing an egg-laying hen;
(3) collecting the immunized eggs, and extracting and purifying yolk antibodies from the yolk of the eggs.
2. The yolk antibody according to claim 1, wherein the step (1) of isolating Vibrio vulnificus from the sample of the Vibrio vulnificus-infected patient comprises the specific steps of: extracting and identifying vibrio vulnificus strain, separating and purifying, culturing and storing.
3. The yolk antibody of claim 2, wherein the operations of extraction, identification, isolation, purification, culture and preservation of Vibrio vulnificus strain are as follows: selecting wound surface blister liquid or tissue exudate of a patient infected with vibrio vulnificus clinically, diluting the wound surface blister liquid or tissue exudate, coating a plate, and culturing overnight; after single colony is picked and centrifugally purified, a mass spectrum analyzer is used for determining whether the colony is infected by the vibrio vulnificus, and the strain determined to be the vibrio vulnificus is subjected to amplification culture.
4. The egg yolk antibody according to claim 1, wherein the preparation of the inactivated bacterial antigen of Vibrio vulnificus in the step (2) comprises the following steps: inactivating the vibrio vulnificus by using a formaldehyde inactivation method to prepare a fully inactivated thallus antigen.
5. The yolk antibody according to claim 1, wherein the specific steps of immunizing laying hens in step (2) are as follows: the antigen solution is injected into the deltoid muscle or pectoralis muscle of the chicken's wing base and inoculated every two weeks.
6. The yolk antibody according to claim 1, wherein the step (2) further comprises measuring the titer of the yolk antibody produced by the immunized laying hens.
7. The yolk antibody according to claim 1, wherein the yolk antibody is extracted and purified from the yolk of the egg in the step (3) by the following steps:
(1) mixing and dissolving yolk water into homogenate, adjusting the pH value of the homogenate to 5.0 +/-0.5, and cooling the acidified homogenate solution to 2-8 ℃ and standing for 1-12 hours;
(2) keeping the temperature of 2-8 ℃ and centrifuging the homogenate at a high speed of 5000-;
(3) adding ammonium sulfate into the supernatant, centrifuging, and collecting precipitate to obtain crude extract;
(4) and (3) redissolving the crude extract in a weak acid buffer solution, and concentrating the redissolved solution through ultrafiltration membranes of 300kD and 100kD in sequence to obtain a yolk antibody mixed concentrated solution.
8. The egg yolk antibody according to claim 7, wherein the mass concentration of ammonium sulfate is 15% -45%.
9. The yolk antibody of claim 1, wherein the step of extracting and purifying the yolk antibody from the yolk of the egg in step (3) further comprises measuring the titer of the yolk antibody and adding a protein stabilizer.
10. Use of the yolk antibody of the inactivated thallus of Vibrio vulnificus according to any one of claims 1 to 9 in the preparation of a reagent or a medicament for preventing or/and treating Vibrio vulnificus infection.
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| CN202010725674.3A CN112175076A (en) | 2020-07-24 | 2020-07-24 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
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| CN202010725674.3A CN112175076A (en) | 2020-07-24 | 2020-07-24 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112175077A (en) * | 2020-08-10 | 2021-01-05 | 中山大学附属第一医院 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
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| CN101343320A (en) * | 2008-07-01 | 2009-01-14 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
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| CN105198988A (en) * | 2015-09-30 | 2015-12-30 | 大连理工大学 | Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
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- 2020-07-24 CN CN202010725674.3A patent/CN112175076A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101343320A (en) * | 2008-07-01 | 2009-01-14 | 华南农业大学 | Anti-vibrio parahaemolyticus chicken yolk antibody, preparation method and application thereof |
| CN104892756A (en) * | 2015-06-18 | 2015-09-09 | 成都安蒂康生物科技有限公司 | Antibacterial chicken egg yolk complex antibody as well as preparation method and application thereof |
| CN105198988A (en) * | 2015-09-30 | 2015-12-30 | 大连理工大学 | Vibrio-splendidus-resistant egg yolk antibody and preparation method thereof |
| CN108003240A (en) * | 2017-12-04 | 2018-05-08 | 西安德轩驰生物科技有限公司 | A kind of multi-joint antiidiotype Yolk antibody vaccine of mariculture fish and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| KASSIM. NEEMA ET AL: "The In Vitro and In Vivo Efficacy of Hen IgY Against Vibrio parahaemolyticus and Vibrio vulnificus", 《J. MICROBIOL. BIOTECHNOL.》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112175077A (en) * | 2020-08-10 | 2021-01-05 | 中山大学附属第一医院 | Preparation method of egg yolk antibody for preventing and treating vibrio vulnificus infection |
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Application publication date: 20210105 |