CN109705214B - Riemerella anatipestifer serum I type and II type egg yolk antibodies and preparation method thereof - Google Patents
Riemerella anatipestifer serum I type and II type egg yolk antibodies and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological medicines, and particularly relates to riemerella anatipestifer serum I type and II type egg yolk antibodies and a preparation method thereof. The method comprises the steps of immunizing laying hens with riemerella anatipestifer to obtain immunized eggs, collecting yolk liquid, and removing impurities through polyacrylamide and caprylic acid to obtain the egg yolk antibody. The riemerella anatipestifer serum I type and II type egg yolk antibodies prepared by the method have high protein content and titer, the cure rate of riemerella anatipestifer is up to more than 90%, and the cured poultry animals can normally grow and develop.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to riemerella anatipestifer serum I type and II type egg yolk antibodies and a preparation method thereof.
Background
Under natural conditions, riemerella anatipestifer disease occurs all the year round. It is mainly infected through contaminated feed, drinking water, dust, spray, etc. through the respiratory tract, digestive tract or wounds of the skin (especially the webbed skin). Ducks of different varieties are likely to be infected and diseased, and particularly, the ducklings of 2-3 weeks old are most susceptible to infection. The occurrence of the disease has a certain relation with the age of the duck, the quality of feeding management, various adverse stress factors or other pathogenic infections, for example, when the sanitary condition is poor, the feeding management is not good, or ducklings are in a conversion environment, the climate is suddenly changed, the ducklings are exposed to cold and rain, and other diseases (muscovy duck hepatopathy, avian colibacillosis, avian rancidity and the like) are mixed and infected, the disease is more easily caused to occur and spread, and the death rate can be up to more than 90%. The disease is difficult to radically cure, the infected ducks are attacked because the disease is early in age of the day and acute in attack, part of ducklings are not immune or do not produce antibodies after the immunization, the ducks with the disease are easy to grow badly and gain slowly, the feeding value is lost, and the disease can be continuously existed in an attack scene, so that the ducks with different batches are easy to infect and attack, and serious economic loss is caused to the duck breeding industry.
Chinese patent application CN107670028A discloses a composite oil emulsion vaccine of riemerella anatipestifer inactivated vaccine and antibody and a preparation method thereof, the vaccine is prepared by three times of emulsification technology of riemerella anatipestifer serum 1, 2 type divalent inactivated vaccine, mineral oil adjuvant and riemerella anatipestifer serum 1, 2 type divalent refined egg yolk antibody, the riemerella anatipestifer inactivated vaccine and the specific antibody thereof are mixed, the problem of blank period of immunity of the traditional inactivated vaccine is successfully solved, the high-efficiency immune effect of effective immunity generated by inoculation is realized, but the method is complex in operation, and the waste of raw materials is easily caused by the combination of the inactivated vaccine and the antibody in the preparation process.
Yolk antibody (IgY) is immunoglobulin present in the yolk of avian animals and is an antibody having a protective effect on offspring produced by transfer of IgG from avian blood to yolk. The molecular mass of the IgY is about 180ku, and the IgY consists of 2 light chains and 2 heavy chains, wherein the molecular mass of the light chains is about 22-30 ku, the molecular mass of the heavy chains is 67-70 ku, and the isoelectric point is close to 5.2. Experiments prove that the IgY concentration in the yolk is maintained at a level equivalent to or higher than the blood concentration for a long time, and the yolk has high stability and good tolerance to heat, acid and alkali environments. The method for producing specific immune antibodies on a large scale by using poultry has the advantages of low cost, high specific antibody titer, good safety, high practicability and easy popularization, and the IgY which is used as a poultry host to produce anti-specific antigens is gradually concerned by scholars.
Chinese patent application CN101475639A discloses a mixed egg yolk antibody for preventing and treating duck infectious serositis and a preparation method thereof, the antibody takes main epidemic serotype strains of Riemerella anatipestifer as antigens, a Riemerella anatipestifer high-immunity egg yolk antibody is prepared, and infection treatment test results show that the protection rate of the prepared egg yolk antibody to serum I type serum II type Riemerella anatipestifer reaches 80% and 90% respectively, the use of antibiotics can be reduced, simultaneously, the quality of livestock products is improved, drug residues are reduced, the safety of human food is promoted, but after the infection of pathogenic bacteria resistant to diseased animals is cured, the growth and development of a part of animals are slowed down, the phenomenon of emaciation and the disease are weak, and the feeding value is lost.
Therefore, the riemerella anatipestifer serum I type and II type egg yolk antibodies which have high cure rate to the riemerella anatipestifer and can normally grow in the cured poultry animals and the preparation method thereof are urgently needed.
Disclosure of Invention
The invention aims to provide riemerella anatipestifer serum I type and II type egg yolk antibodies which have high cure rate on riemerella anatipestifer and can normally grow on cured poultry animals and a preparation method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a riemerella anatipestifer serum I type and II type egg yolk antibody is prepared by the following steps:
s1, immunizing the laying hens with the riemerella anatipestifer serum I type and II type inactivated vaccines, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
s2, mixing the yolk solution obtained in the step S1 with a Tris-HCl buffer salt solution, adding a polyacrylamide solution, uniformly mixing, standing at 1-4 ℃ for 1-4 h, and centrifuging at 2000-5000 rpm for 10-15 min to obtain a supernatant;
s3, adding the n-octanoic acid solution into the supernatant obtained in the step S2, uniformly mixing, standing, centrifuging at 2000-5000 rpm for 20-30 min, extracting the lower layer clear liquid, filtering, concentrating, and performing aseptic treatment to obtain the finished product.
Further, in the step S1, the weight ratio of the Riemerella anatipestifer serum I type and the II type inactivated vaccine is (1-3): 1.
Further, in the step S1, the ratio by weight of the riemerella anatipestifer serum I type and II type inactivated vaccine is 3: 1.
Further, in the step S2, the yolk solution and the Tris-HCl buffer salt are mixed according to the volume ratio of 1 (2-5).
Further, in step S2, the polyacrylamide solution is added to make the final weight fraction of polyacrylamide be 0.1-2%.
Further, in step S2, the polyacrylamide solution is added to make the final weight fraction of polyacrylamide be 1%.
Further, in step S3, the n-octanoic acid solution is added to make the final weight fraction of n-octanoic acid be 0.1-0.3%.
Further, in step S3, the n-octanoic acid solution is added to make the final weight fraction of n-octanoic acid 0.2%.
In addition, the invention also provides a preparation method of the yolk antibody, which comprises the following steps:
A. immunizing laying hens with riemerella anatipestifer serum I-type and II-type inactivated vaccines, wherein the weight ratio of the riemerella anatipestifer serum I-type to the II-type inactivated vaccines is (1-3): 1, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
B. mixing the yolk solution obtained in the step A with a Tris-HCl buffer salt solution according to a volume ratio of 1 (2-5), adding a polyacrylamide solution to enable the final weight fraction of polyacrylamide to be 0.1-2%, uniformly mixing, standing for 1-4 h at 1-4 ℃, and centrifuging for 10-15 min at 2000-5000 rpm to obtain a supernatant;
C. and C, adding an n-caprylic acid solution into the supernatant obtained in the step B to enable the final weight fraction of the n-caprylic acid to be 0.1-0.3%, uniformly mixing, standing, centrifuging at 2000-5000 rpm for 20-30 min, extracting a lower layer of clear liquid, filtering, concentrating and carrying out aseptic treatment to obtain the n-caprylic acid.
In addition, the invention also provides application of the egg yolk antibody in preparation of a medicament for preventing and treating riemerella anatipestifer or poultry feed.
According to the invention, the laying hens are immunized by the riemerella anatipestifer serum I type and II type inactivated vaccines in a certain proportion, the obtained yolk liquid is subjected to impurity removal by using a certain amount of polyacrylamide and n-caprylic acid, the finally prepared egg yolk antibody has high protein content and high titer, the cure rate for the riemerella anatipestifer is high, and the cured poultry animals can normally grow and develop. Test example 1 can prove that the yolk antibody prepared in the examples 1-3 is colorless, high in clarity, and high in protein content and potency; experimental example 2 can prove that the protection rate of the egg yolk antibody prepared in the embodiments 1-3 on riemerella anatipestifer is more than 90%, and the growth and development of the protected ducklings are normal after 14 and 30 days. Although the egg yolk antibodies prepared in the comparative examples 1 to 3 can protect ducklings attacked by bacteria to a certain extent, 30 to 40 percent of the ducklings are dysplastic and relatively thin after 14 to 30 days, and 10 to 20 percent of the ducklings are gradually killed, so that the final protection rate is only 50 to 70 percent.
The invention has the following advantages:
(1) the riemerella anatipestifer serum I type and II type egg yolk antibodies have high protein content and high titer, the cure rate of the riemerella anatipestifer is up to more than 90 percent, and the cured poultry animals can normally grow and develop.
(2) The preparation method of the riemerella anatipestifer serum I type and II type egg yolk antibodies has simple operation steps, is suitable for large-scale production of the riemerella anatipestifer serum I type and II type egg yolk antibodies, and reduces the economic loss of poultry breeding industry.
Detailed Description
The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.
The preparation method of the riemerella anatipestifer serum I-type and II-type inactivated vaccines comprises the following steps:
taking Riemerella anatipestifer serum I type (RA1) and Riemerella anatipestifer blood II type (RA2) strains, respectively inoculating the strains into tryptone yeast broth containing 0.5% serum, performing shake culture at 37 ℃ to logarithmic growth phase, centrifuging to obtain precipitates, dissolving the precipitates in PBS buffer solution, centrifuging to collect thalli, inactivating the thalli by using methanol for 36 hours, and removing the methanol to obtain the inactivated vaccine. The Riemerella anatipestifer serum I type (RA1) and Riemerella anatipestifer blood II type (RA2) strains are provided by southern China agricultural university.
The other reagents are common reagents and can be purchased from conventional reagent production and sale companies.
Example 1 Riemerella anatipestifer serotype I and II yolk antibodies
The preparation method comprises the following steps:
A. immunizing laying hens with riemerella anatipestifer serum I type and II type inactivated vaccines in a weight ratio of 3:1, adding pharmaceutically-acceptable adjuvants to prepare an injection with 50% of inactivated vaccine content, wherein the dose is 1 ml/egg, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
B. mixing the yolk solution obtained in the step A with a Tris-HCl buffer salt solution according to a volume ratio of 1:3, adding a polyacrylamide solution to enable the final weight fraction of polyacrylamide to be 1%, uniformly mixing, standing at 4 ℃ for 2h, and centrifuging at 3000rpm for 10min to obtain a supernatant;
C. and C, adding an n-caprylic acid solution into the supernatant obtained in the step B to enable the final weight fraction of the n-caprylic acid to be 0.2%, uniformly mixing, standing, centrifuging at 4000rpm for 20min, extracting a lower layer of clear liquid, filtering, concentrating, and performing aseptic treatment to obtain the n-caprylic acid.
Example 2 Riemerella anatipestifer serum type I and type II yolk antibodies
The preparation method comprises the following steps:
A. immunizing laying hens with riemerella anatipestifer serum I type and II type inactivated vaccines, wherein the weight ratio of the riemerella anatipestifer serum I type to the II type inactivated vaccines is 1:1, adding pharmaceutically-allowable adjuvants to prepare an injection with the content of the inactivated vaccines being 50%, the dosage being 1 ml/egg, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
B. mixing the yolk solution obtained in the step A with a Tris-HCl buffer salt solution according to a volume ratio of 1:4, adding a polyacrylamide solution to enable the final weight fraction of polyacrylamide to be 0.5%, uniformly mixing, standing at 4 ℃ for 1h, and centrifuging at 3000rpm for 15min to obtain a supernatant;
C. and C, adding an n-caprylic acid solution into the supernatant obtained in the step B to enable the final weight fraction of the n-caprylic acid to be 0.1%, uniformly mixing, standing, centrifuging at 5000rpm for 20min, extracting a lower layer of clear liquid, filtering, concentrating, and performing aseptic treatment to obtain the n-caprylic acid.
Example 3 Riemerella anatipestifer serotype I and II yolk antibodies
The preparation method comprises the following steps:
A. immunizing laying hens with riemerella anatipestifer serum I type and II type inactivated vaccines in a weight ratio of 2:1, adding pharmaceutically-acceptable adjuvants to prepare an injection with 50% of inactivated vaccine content, wherein the dose is 1 ml/egg, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
B. mixing the yolk solution obtained in the step A with a Tris-HCl buffer salt solution according to a volume ratio of 1:5, adding a polyacrylamide solution to enable the final weight fraction of polyacrylamide to be 2%, uniformly mixing, standing at 4 ℃ for 3h, and centrifuging at 4500rpm for 10min to obtain a supernatant;
C. and C, adding an n-caprylic acid solution into the supernatant obtained in the step B to enable the final weight fraction of the n-caprylic acid to be 0.3%, uniformly mixing, standing, centrifuging at 4500rpm for 30min, extracting a lower layer of clear liquid, filtering, concentrating, and performing aseptic treatment to obtain the n-caprylic acid.
Comparative example 1 yolk antibody
Except for example 1, comparative example 1 replaced polyacrylamide in step B with poloxamer, and the remaining parameters and operations refer to example 1.
Comparative example 2 yolk antibody
The difference from example 1 is that the final weight fraction of polyacrylamide in step B of comparative example 2 is 3.5%, and the rest of the parameters and operation refer to example 1.
Comparative example 3 yolk antibody
The difference from example 1 is that the final weight fraction of n-octanoic acid in step C of comparative example 2 is 0.4%, and the rest of the parameters and operation refer to example 1.
Test example 1 measurement of protein concentration and titer
The protein content of the egg yolk antibodies prepared in examples 1 to 3 and comparative examples 1 to 3 was measured by a NanoDrop Spectrophotometer ND-1000 type ultraviolet Spectrophotometer, and the antibody titer was measured by a conventional agar diffusion method, and the results are shown in Table 1.
TABLE 1 results of measurement of egg yolk antibody protein concentration and titer
| Group of | Clarity of the product | Colour(s) | Protein concentration | Potency of the drug |
| Example 1 | ++++ | Colorless and colorless | 8.45 | 24 |
| Example 2 | ++++ | Colorless and colorless | 8.41 | 24 |
| Example 3 | ++++ | Colorless and colorless | 8.32 | 24 |
| Comparative example 1 | ++++ | Colorless and colorless | 8.29 | 24 |
| Comparative example 2 | ++++ | Colorless and colorless | 7.63 | 23 |
| Comparative example 3 | ++++ | Colorless and colorless | 7.58 | 23 |
As can be seen from Table 2, the yolk antibodies prepared in the examples 1 to 3 of the invention are colorless, have high clarity, and have high protein content and potency, while the protein concentration and the potency of the comparative examples 2 to 3 (increasing the content of polyacrylamide or caprylic acid, respectively) are significantly reduced.
Test example 2 yolk antibody infection protection test
1. Test materials: the yolk antibodies prepared in examples 1 to 3 and comparative examples 1 to 3.
2. Test subjects: 5-10 days old ducklings.
3. The test method comprises the following steps:
140 ducklings with the age of 5-10 days are randomly divided into groups of examples 1-3, groups of comparative examples 1-3 and blank controls 1 and 2, and RA1 and RA2 are used for attacking the ducklings respectively, and the attacking is carried out by adopting a neck subcutaneous injection method, wherein the dose is 0.5 ml/ducklings (the bacteria content is 2 hundred million). After 36 hours of challenge, 2 ml/each of the groups was injected with the corresponding antibody, and 1 group of RA1 and RA2 was simultaneously injected with an equal amount of physiological saline without treatment, and used as a blank control 1 group and a blank control 2 group. Animals of each group were observed for morbidity, mortality, and growth on days 7, 14, and 30.
4. And (3) test results:
TABLE 2 infection protection test results (% survival)
As can be seen from table 2, within 7 days after challenge, all the ducklings of the blank control group 1 and the blank control group 2 were all attacked or dead, and severe pericarditis and liver or pleural adhesions were observed by autopsy. The yolk antibody prepared in the examples 1-3 can ensure that most of ducklings survive, the protection rate is over 90 percent, and the growth and development of the ducklings after 14 days and 30 days are normal. Although the egg yolk antibodies prepared in the comparative examples 1 to 3 can protect ducklings attacked by bacteria to a certain extent, 30 to 40 percent of the ducklings are dysplastic and relatively thin after 14 to 30 days, and 10 to 20 percent of the ducklings are gradually killed, so that the final protection rate is only 50 to 70 percent.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (4)
1. A preparation method of riemerella anatipestifer serum I type and II type egg yolk antibodies is characterized by comprising the following steps:
A. immunizing laying hens with riemerella anatipestifer serum I-type and II-type inactivated vaccines, wherein the weight ratio of the riemerella anatipestifer serum I-type to the II-type inactivated vaccines is (1-3): 1, collecting eggs at 7 days after immunization, and separating proteins after disinfection to obtain yolk liquid;
B. mixing the yolk solution obtained in the step A with a Tris-HCl buffer salt solution according to a volume ratio of 1 (2-5), adding a polyacrylamide solution to enable the final weight fraction of polyacrylamide to be 0.1-2%, uniformly mixing, standing for 1-4 h at 1-4 ℃, and centrifuging for 10-15 min at 2000-5000 rpm to obtain a supernatant;
C. and C, adding an n-caprylic acid solution into the supernatant obtained in the step B to enable the final weight fraction of the n-caprylic acid to be 0.1-0.3%, uniformly mixing, standing, centrifuging at 2000-5000 rpm for 20-30 min, extracting a lower layer of clear liquid, filtering, concentrating and carrying out aseptic treatment to obtain the n-caprylic acid.
2. The method for preparing a yolk antibody according to claim 1, wherein in the step A, the weight ratio of the riemerella anatipestifer inactivated vaccine of type I and type II is 3: 1.
3. The method for producing a yolk antibody according to claim 1, wherein in the step B, the polyacrylamide solution is added so that the final weight fraction of polyacrylamide is 1%.
4. The method for producing a yolk antibody according to claim 1, wherein in the step C, the solution of n-caprylic acid is added so that the final weight fraction of n-caprylic acid is 0.2%.
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| CN102716483A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Duck plague yolk antibody freeze-dried powder and preparation method thereof |
| CN108753811A (en) * | 2018-06-29 | 2018-11-06 | 山东省农业科学院家禽研究所 | A kind of recombinant plasmid, structure, expression and application for riemerella anatipestifer novel subunit vaccine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102716483A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Duck plague yolk antibody freeze-dried powder and preparation method thereof |
| CN108753811A (en) * | 2018-06-29 | 2018-11-06 | 山东省农业科学院家禽研究所 | A kind of recombinant plasmid, structure, expression and application for riemerella anatipestifer novel subunit vaccine |
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| 鸭卵黄免疫球蛋白IgY研究进展;袁涛等;《现代畜牧兽医》;20060215;第52页右栏第2.2部分 * |
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