CN112679606A - Hyperimmunity serum of erysipelothrix rhusiopathiae and preparation method thereof - Google Patents
Hyperimmunity serum of erysipelothrix rhusiopathiae and preparation method thereof Download PDFInfo
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Abstract
The invention provides a swine erysipelas high-immunity serum and a preparation method thereof, the high-immunity serum is obtained by immunizing a swine erysipelas inactivated vaccine prepared by taking a swine erysipelas type 1a strain as an antigen and matching with a traditional Chinese medicine immunopotentiator, wherein the traditional Chinese medicine immunopotentiator is one or more of ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide. The traditional Chinese medicine immunopotentiator in the hyperimmune serum of the erysipelothrix rhusiopathiae can enhance the disease resistance of organisms and improve the antibody level of the organisms, and the preparation method has the advantages of simple operation, low cost and capability of obtaining large amount of hyperimmune serum.
Description
Technical Field
The invention belongs to the field of veterinary biotechnology, and particularly relates to erysipelothrix rhusiopathiae hyperimmune serum and a preparation method thereof.
Background
Erysipelothrix rhusiopathiae (Erysipelothrix rhusiopathiae), commonly known as Erysipelothrix rhusiopathiae, is widely distributed in nature and can cause erysipelas in pigs and other animals. In a swinery, swine erysipelas caused by swine erysipelas is an acute and febrile infectious disease and is clinically manifested as acute septicemia, subacute rash type and chronic endocarditis type. The erysipelothrix rhusiopathiae is divided into 26 serotypes including 1a type, 1b type, 2-24 type and N type, and most of the erysipelothrix rhusiopathiae separated from the body of the pig with acute septicemia is 1a type. Before the 80's in the 20 th century, swine erysipelas was one of three infectious diseases (swine fever, swine erysipelas and swine plague) which endanger the swine industry in China. Later, with the development of the breeding industry in China and the use of vaccines and antibiotics, few reports about swine erysipelas cases exist from the later 90 s of the 20 th century to the 21 st century. However, since 2010, the swine erysipelas is sporadically popular in a plurality of provinces of China, mainly occurs in large-scale pig farms, the incidence rate also tends to rise year by year, serious economic loss is caused to the pig raising industry of China, and meanwhile, the swine erysipelas poses certain threats to human health.
The acute swine erysipelas is acute in morbidity and the mortality rate can reach 80% -90%, so that the treatment of the acute swine erysipelas is particularly important, and the acute swine erysipelas can be treated symptomatically by adopting hyper-immune serum and antibiotics to obtain a better curative effect. In addition, hyperimmune serum can be used to identify serotypes of erysipelothrix rhusiopathiae.
Disclosure of Invention
In view of the above, the invention provides a hyperimmune serum of erysipelothrix rhusiopathiae and a preparation method thereof, aiming at overcoming the defects in the prior art.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a hyperimmune serum of Erysipelothrix rhusiopathiae is prepared by immunizing inactivated vaccine of Erysipelothrix rhusiopathiae prepared from type 1a strain as antigen and Chinese medicinal immunopotentiator, wherein the Chinese medicinal immunopotentiator is one or more of Ginseng radix polysaccharide, radix astragali polysaccharide, radix Angelicae sinensis polysaccharide, and herba Epimedii polysaccharide.
Further, the traditional Chinese medicine immunopotentiator is ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide to the angelica polysaccharide to the epimedium polysaccharide is (2-3):1:1: 1.
preferably, the traditional Chinese medicine immunopotentiator is ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide, and the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide to the angelica polysaccharide to the epimedium polysaccharide is 2:1:1: 1.
further, the mass ratio of the antigen to the traditional Chinese medicine immunopotentiator is 4: 1.
further, the swine erysipelas inactivated vaccine also comprises an adjuvant, wherein the adjuvant is a mineral oil adjuvant or Freund's adjuvant.
Furthermore, the swine erysipelas inactivated vaccine also comprises a preservative, and the preservative is thimerosal or phenol.
Furthermore, in the swine erysipelas inactivated vaccine, the content of the inactivated thallus of the swine erysipelas 1a type strain is not less than 3.0 multiplied by 109CFU/mL。
The invention also provides a preparation method of the erysipelothrix rhusiopathiae hyperimmune serum, which specifically comprises the following steps:
1) culturing erysipelothrix rhusiopathiae type 1a strain and inactivating the strain liquid;
2) concentrating the inactivated bacterial liquid, removing supernatant, washing and diluting to obtain antigen liquid;
3) mixing the antigen solution with traditional Chinese medicine immunopotentiator, adjuvant and antiseptic uniformly to obtain swine erysipelas inactivated vaccine;
4) carrying out 1 primary immunization on piglets by using the swine erysipelas inactivated vaccine, carrying out primary boosting immunization after 21d, carrying out secondary boosting immunization at an interval of 21d, carrying out primary challenge by using the swine erysipelas 1a type strain after 14d of immunization, and carrying out secondary challenge after 14d of interval;
5) collecting blood, separating serum, testing antibody titer to 210Bleeding all the carotid arteries of the piglets;
6) placing the collected blood in a constant temperature incubator at 37 ℃ for 1h, and then placing the blood in a refrigerator at 4 ℃ for 12-16 h; centrifuging the collected blood at 3000r/min for 5min, separating supernatant to obtain serum, filtering with 0.22 μm filter membrane, and packaging to obtain hyperimmune serum of Erysipelothrix rhusiopathiae;
7) the antibody level in the serum is detected by using a thallus ELISA method, and the titer reaches 210The hyperimmune serum of the erysipelothrix rhusiopathiae is obtained.
Further, in the step 1), the content of viable bacteria of the erysipelothrix rhusiopathiae type 1a strain in the bacterial liquid is not less than 1.0 multiplied by 109CFU/mL。
Further, in the step 1), formaldehyde or beta-propiolactone is used for inactivation.
Further, in the step 1), a formaldehyde inactivating agent is adopted for inactivation, and a formaldehyde solution with the mass concentration of 37% -40% is added into the formaldehyde inactivating agent according to 0.25% -0.3% of the total volume fraction of the bacterial liquid; inactivating at 37-39 deg.C for 24-48 h.
Further, in the step 2), sterile PBS solution with the concentration of 0.0l mol/L, pH value of 7.2 can be adopted for washing and diluting; after washing, the bacterial antigens were diluted with sterile PBS.
Further, in the step 3), the traditional Chinese medicine immunopotentiator is obtained by mixing ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide according to the mass ratio of (2-3) to 1:1:1, and the antigen solution and the traditional Chinese medicine immunopotentiator are mixed according to the mass ratio of 4: 1.
Further, the adjuvant in the step 3) is a mineral oil adjuvant, the antigen liquid added with the traditional Chinese medicine immunopotentiator and the mineral oil adjuvant are mixed according to the volume ratio of 1:2, and emulsified for 5-10min at room temperature of 8000-.
Further, the adjuvant in the step 3) is Freund's adjuvant, and the antigen solution added with the traditional Chinese medicine immunopotentiator is mixed with the Freund's adjuvant according to the volume ratio of 1: 1; emulsifying at 8000-.
Further, in the step 3), the preservative is thimerosal, and the addition amount of the preservative is 0.005% of the volume of the antigen solution.
Further, in step 3), the preservative is phenol, and the added amount thereof is 0.3% by volume of the antigen liquid.
Further, in the step 3), the content of the inactivated erysipelas suis type 1a strain in the inactivated erysipelas suis vaccine is not less than 3.0 multiplied by 109CFU/mL。
Further, in step 4), the immunization route is neck muscle immunization, and the dosage is 2 mL/head.
Further, in the step 7), the antibody detection method is a thallus ELISA detection antibody, namely, the inactivated thallus is crushed to be clear, then the thallus is centrifuged at 12000r/min for 10min, cell fragments are discarded, and the supernatant is an antigen for ELISA detection.
The preparation of the hyperimmune serum is usually prepared by vaccine immunization, the effect of the vaccine is the key of the high or low antibody titer in the hyperimmune serum, the effect of the vaccine can be influenced by various factors, and the method for improving the immune efficacy of the vaccine by adding the traditional Chinese medicine immunopotentiator into the vaccine is a method with good effect and small toxic and side effects. In the early stage, ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide are screened from a large amount of traditional Chinese medicine immunopotentiators and are added into a swine erysipelas inactivated vaccine to enhance the immunity. The panaxan can enhance phagocytosis of phagocyte, increase antibody production ability, and stimulate interferon production. The astragalus polysaccharide can participate in humoral immunity, promote the transformation of lymphocytes, enhance the immunity of organisms and improve the growth performance of piglets. The angelica polysaccharide can activate T, B lymphocytes, macrophages, natural killer cells and other immune cells, can promote the generation of cytokines, activate a complement system, promote the generation of antibodies and play a multi-aspect regulating role on the immune system. The herba Epimedii polysaccharide has effects of regulating immunity, resisting virus, and stimulating antibody generation. The inventor of the application innovatively and unexpectedly finds that the serum antibody titer obtained by using the four polysaccharides in combination as the traditional Chinese medicine immunopotentiator is high, and a large amount of hyperimmune serum can be obtained.
Compared with the prior art, the invention has the following advantages:
the invention also provides a preparation method of the hyperimmune serum with high titer, simple operation, low cost and large obtained amount, which is used for matching the treatment of acute swine erysipelas and the serotype identification of swine erysipelas.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention will be described in detail with reference to the following examples.
Examples
The preparation of the swine erysipelas inactivated vaccine by using the swine erysipelas mycelium 1a type strain comprises the following steps:
1. seed preparation for production
1) First order seed reproduction
Inoculating the erysipelothrix rhusiopathiae type 1a strain production strain on a TSA culture medium containing 10% newborn bovine serum, culturing at the constant temperature of 37 ℃ for 36h, then selecting more than 5 typical colonies, respectively inoculating the typical colonies on the TSA culture medium containing 10% newborn bovine serum, culturing at the temperature of 37 ℃ for 24h, and taking the obtained product as a first-stage seed after passing a pure inspection. The product is stored at the temperature of 2-8 ℃, the storage time is not more than 7 days, and the subculture is not more than 5 generations.
2) Two stage seed reproduction
Inoculating the strain primary seeds into a TSB liquid culture medium containing 10% newborn calf serum, placing the strain primary seeds on a shaker for shaking culture at 37 ℃ and 180r/min for 14h, and using the strain primary seeds as secondary seeds after passing inspection. The product is stored at the temperature of 2-8 ℃, and the service life is not more than 7 days.
2. Bacterial liquid culture
And (2) filling a proper amount of TSB liquid culture medium containing 10% newborn calf serum into a glass bottle according to the volume, then transferring the secondary seeds into the new TSB culture medium according to the proportion of 1:100, and performing shaking culture at 37 ℃ and 180r/min for 16h to obtain bacterial liquid.
3. Viable count
Viable bacteria count was carried out using a TSA culture medium containing 10% newborn bovine serum suitable for growth of the present bacteria according to the method in appendix of the existing "Chinese veterinary pharmacopoeia". Cultured in a bacteria bottle, the content of viable bacteria of type 1a strain of Erysipelothrix rhusiopathiae is not less than 1.0 × 109CFU/mL。
4. Inactivation of bacterial liquid
The culture solution of the erysipelothrix rhusiopathiae type 1a strain is inactivated, formaldehyde solution (with the concentration of 37-40%) is added according to 0.3% (volume ratio) of the total amount of the bacterial solution, and the bacterial solution is inactivated for 48 hours at 37 ℃.
5. Inactivation test
Inoculating 0.2mL of inactivated bacterial liquid into a TSA culture medium (containing 10% newborn calf serum) suitable for the growth of the bacteria, inoculating 0.2mL of inactivated bacterial liquid into a TSB liquid culture medium (containing 10% newborn calf serum) suitable for the growth of the bacteria according to the proportion of 1:100, and continuously culturing at 37 ℃ for 7 days until the bacteria grow aseptically, thus obtaining the qualified product.
6. Seedling preparation
1) Preparation of antigens
And (3) concentrating the bacterial liquid qualified by inactivation inspection by using a hollow fiber ultrafilter to remove the supernatant (or centrifuging the bacterial liquid qualified by inactivation inspection by using a low-temperature high-speed centrifuge to remove the supernatant), washing the bacterial liquid by using sterile PBS (0.0l mol/L, pH value of 7.2), and diluting the bacterial antigen by using the sterile PBS (0.0l mol/L, pH value of 7.2) according to the counting result of viable bacteria before inactivation to prepare antigen suspension.
2) Seedling preparation
Oil phase: adding span-80 and aluminum stearate into mineral oil at a ratio of 93:6:1, mixing, autoclaving at 121 deg.C for 30min, and cooling.
Water phase: diluting the concentrated antigen with sterile PBS (0.0L mol. L-1, pH 7.2) to obtain antigen suspension, adding sterilized Tween-80 with final concentration of 4% into the antigen suspension, and stirring until Tween-80 is completely dissolved (while stirring) to obtain water phase-1; mixing the antigen suspension added with tween with Ginseng radix polysaccharide, radix astragali polysaccharide, radix Angelicae sinensis polysaccharide and herba Epimedii polysaccharide (the addition ratio of each component is 1:1:1:1) at a ratio of 4:1 to obtain water phase-2; mixing the antigen suspension with tween, Ginseng radix polysaccharide, radix astragali polysaccharide, radix Angelicae sinensis polysaccharide and herba Epimedii polysaccharide (the addition ratio of each component is 2:1:1:1) at a ratio of 4:1 to obtain water phase-3.
And (3) emulsification preparation of the vaccine: the ratio of oil phase to water phase added was 2:1 (i.e. 2 parts oil phase, 1 part water phase). Adding 2 parts of oil phase into a stirrer, starting a motor to stir at a low speed (30s), slowly adding 1 part of water phase, emulsifying at 8000-10000r/min for 5-10min, adding thimerosal in an amount of 0.005% (volume percentage) before stopping stirring, and fully and uniformly mixing. Finally, the content of inactivated thallus in the vaccine is not less than 3.0 multiplied by 109CFU/mL。
3) Inspection of semi-finished product
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the result shows that the bacteria-free growth is realized.
4) Dispensing
Quantitatively subpackaging, covering and sealing, and storing at 2-8 ℃.
Test example 1
In the examples, the finished product of the swine erysipelas inactivated vaccine is tested:
1) traits
The product is white homogeneous emulsion, is in oil-drop shape without diffusion when dropped into water, and has no layering and demulsification when placed at 4 ℃ for 3 months.
2) Load check
The inspection is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are met.
3) Sterility testing
The test is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the sterile growth is carried out.
4) Safety inspection
Safety test for piglet vaccination: 9 batches (3 batches each) of swine erysipelas inactivated vaccines (batch numbers: 202001, 202002, 202003, 202004, 202005, 202006, 202007, 202008 and 202009) which are pilot-produced according to the method of example 2 are respectively used for 2-fold dose inoculation (4 mL/head) on healthy piglets of 3-4 weeks old, meanwhile, a control group is established for inoculation of sterile PBS (0.0l mol/L, pH value 7.2) (4 mL/head), the immunization part is neck muscle, and clinical symptoms and pathological changes of the piglets are closely observed after immunization.
The results show that 9 batches of vaccinated piglets have no local or systemic adverse reactions such as mental state, appetite abnormality, vomit, diarrhea and even death and the like caused by vaccination. The results of body temperature measurement before and one week after inoculation show that the piglets in the immune group have transient body temperature rise, but the average body temperature rise at 24h after inoculation does not exceed 1 ℃, and the piglets are recovered to be normal within 48 h. After the observation period is finished, the immune parts of the piglets of each group of the experimental piglets are anatomically observed, the immune parts of the piglets of 9 batches of the vaccine groups have no obvious phenomena of vaccine residue, local inflammation, tissue lesion and the like, and no granuloma is formed, and the differences are not obvious compared with a control group. The test results show that the swine erysipelas inactivated vaccine in the embodiment is safe to inoculate target animals (piglets at 3-4 weeks old) with minimum days old.
5) Measurement of residual Formaldehyde and Thimerosal
The determination is carried out according to the appendix of the current Chinese veterinary pharmacopoeia, and the regulations are met.
Test example 2
In the examples, the potency test and antibody titer test of three finished products of swine erysipelas inactivated vaccines are as follows:
1) immunization and toxin counteracting for piglet
25 healthy piglets of 4-5 weeks old are randomly divided into 5 groups (5 piglets/group), and the specific grouping conditions are shown in table 1. Vaccine 1 group was immunized with 2mL of vaccine injected intramuscularly in each neck of each piglet, and after 21 days of immunization, 2mL of the same vaccine was inoculated according to the same method for boosting. The vaccine 2 group and the vaccine 3 group respectively immunize the vaccine of which the adding proportion of each component of the traditional Chinese medicine immunopotentiator is 1:1:1:1 and the vaccine of which the adding proportion of each component of the traditional Chinese medicine immunopotentiator is 2:1:1:1, and after the vaccine is immunized for 21 days respectively, the vaccines are immunized according to the same method2mL of the same vaccine was inoculated for booster immunization. Control groups were inoculated with 2mL of sterile PBS each time as blank control. The piglets of the healthy group are not inoculated as healthy control groups and are fed separately without attacking poison. After the second immunization 14d, the vaccine group and the control group were injected with a 5 LD-containing solution via the ear-edge vein501mL of the bacterial liquid of the parent strain of erysipelothrix rhusiopathiae type 1a (the number of viable bacteria contained in the bacterial liquid is 1.6 multiplied by 10)5CFU), all piglets were observed daily for morbidity to day 14. The results are shown in Table 1.
TABLE 1 immunoprotection Effect of piglets in different immunization groups
Note: "-" indicates inapplicable.
2) Serum separation and antibody titer detection
Before first immunization, after first immunization and after second immunization, jugular vein blood collection and serum separation are respectively carried out on each group of piglets.
The erysipelothrix rhusiopathiae type 1a strain is inoculated on a TSA solid culture medium containing 10% newborn bovine serum, the culture is carried out at the constant temperature of 37 ℃ for 24-48h, and then a single colony is picked by an inoculating loop and is subjected to purification culture for 18-24 h. The cultured single colony is picked up and cultured in a TSB liquid culture medium containing 10% newborn calf serum for 14h at 37 ℃ as a seed solution, then the seed solution is transferred into a new TSB liquid culture medium (containing 10% newborn calf serum) according to the proportion of 1:100, and the shaking culture is carried out for 16h at 37 ℃ and 180 r/min. Adding 0.3% formaldehyde solution into the cultured bacterial liquid for inactivation for 48h, centrifuging the inactivated bacterial liquid at 4 ℃, discarding the supernatant, collecting the bacterial body, and washing the bacterial body for 3 times by using sterile PBS buffer solution. And (3) concentrating the washed thallus according to the ratio of 1:5, and carrying out heavy suspension by using a sterile PBS buffer solution to obtain thallus suspension. And (3) crushing the thallus suspension according to the instructions of an ultrasonic crusher until the liquid is clear, then centrifuging at 12000r/min for 10min, removing cell fragments, and subpackaging the supernatant into an EP tube and placing at 4 ℃ for later use.
Adding 180 mu l of coating solution into each hole of the first row (from bottom to top) of a 96-hole enzyme-linked reaction plate (12 holes in each row and 8 holes in each column), adding 100 mu l of coating solution into each hole of the other rows, adding 20 mu l of prepared antigen into each hole of the first row, sucking 100 mu l of prepared antigen upwards from the first row after blowing, uniformly mixing, diluting by times, discarding 100 mu l in the last row, and coating 1 row in each concentration. Then left at 37 ℃ for 1h and then at 4 ℃ overnight, taken out and washed 3 times with washing solution (3 min each). Add 100. mu.l of blocking solution to each well, leave at 37 ℃ for 1h, take out and wash 3 times with wash solution (3 min each). Then, 180. mu.l of blocking solution was added to each well of the first column (from left to right) of the new 96-well enzyme-linked reaction plate, 100. mu.l of blocking solution was added to each well of the remaining columns, 20. mu.l of serum was added to each well of the first column, and 100. mu.l of serum was aspirated from the first column by a pipette and diluted to the right, i.e., one column was diluted at each concentration. 100. mu.l of diluted serum was removed from each well and added to the corresponding coated plate well, left at 37 ℃ for 30min, and washed 5 times (3 min each) with washing solution after removal. Then, 100. mu.l of goat anti-porcine IgG-HRP diluted with blocking solution (ratio: 5000) was added to each well, and the mixture was left at 37 ℃ for 30min, and then washed with washing solution 5 times (3 min each time). Adding 100 mul of TMB color development liquid into each hole, developing for 10min in a dark place, and then adding 100 mul of TMB reaction termination liquid into each hole. The sample was placed in a microplate reader and OD value was measured at a wavelength of 450 nm.
Add 100 μ l of antigen diluted according to the optimal coating concentration to each well of a 96-well enzyme-linked reaction plate, put at 37 ℃ for 1h and then overnight at 4 ℃, take out and wash with washing solution 3 times (3 min each time), add 100 μ l of blocking solution to each well, stand at 37 ℃ for 1h, take out and wash with washing solution 3 times (3 min each time). Add 100. mu.l of serum to be tested (diluted with blocking solution according to optimal dilution) to each well of the coated plate, leave at 37 ℃ for 30min, and wash 5 times (3 min each time) with washing solution. Then, 100. mu.l of goat anti-porcine IgG-HRP diluted with blocking solution (ratio: 5000) was added to each well, and the mixture was left at 37 ℃ for 30min, and then washed 5 times with washing solution (3 min each time) after being taken out. Adding 100 μ l of TMB color developing solution into each well, developing for 10min in dark place, and adding 100 μ l of TMB reaction termination solution into each well. The OD value was measured at a wavelength of 450nm in a microplate reader, and the results are shown in Table 2.
TABLE 2 antibody levels in sera of piglets of different immunization groups
| Group of | OD value | Antibody titer |
| Vaccine 1 group | 0.533 | 23 |
| Vaccine 2 group | 0.765 | 25 |
| Vaccine 3 groups | 0.885 | 26 |
| Control group | 0.142 | —— |
Note: "-" indicates inapplicable.
Tests prove that the immune protection effect of the vaccine group added with the traditional Chinese medicine immunopotentiator is obviously stronger than that of the vaccine group without the traditional Chinese medicine immunopotentiator, and the vaccine group added with the ginseng polysaccharide, the astragalus polysaccharide, the angelica polysaccharide and the epimedium polysaccharide (the adding ratio of each component is 2:1:1:1) has stronger immune protection effect and higher antibody titer in serum, so that the vaccine added with the ginseng polysaccharide, the astragalus polysaccharide, the angelica polysaccharide and the epimedium polysaccharide (the adding ratio of each component is 2:1:1:1) is selected to prepare the high immune serum.
Test example 3
According to the results of the experimental example 2, the swine erysipelas inactivated vaccine added with the ginseng polysaccharide, the astragalus polysaccharide, the angelica polysaccharide and the epimedium polysaccharide (the adding ratio of each component is 2:1:1:1) in the example is used for preparing the hyperimmune serum.
1) Immunization and toxin counteracting for piglet
20 healthy piglets of 4-5 weeks old are randomly divided into 4 groups (5 piglets/group), and the specific grouping conditions are shown in table 3. The group 1 is a vaccine immunization group, 2mL of vaccine is injected into each neck of each piglet through muscle for basic immunization, and after 21d and 42d of basic immunization, 2mL of vaccine is inoculated according to the same method for twice boosting immunization. Piglets in groups 2 and 3 were not vaccinated with 2mL sterile PBS each time as a blank control group. Piglets in group 4 were not inoculated as a healthy control group, and were kept in isolation without counteracting toxic substances. After 14d of booster immunization, groups 1 and 2 were injected intravenously via the ear margin with approximately 5LD501mL of the bacterial liquid of the parent strain of erysipelothrix rhusiopathiae type 1a (the number of viable bacteria contained in the bacterial liquid is 1.2 multiplied by 10)5CFU), all piglets were observed daily for 14 days, and then group 1 and group 3 were injected with 10LD by ear vein501mL of the bacterial liquid of the parent strain of erysipelothrix rhusiopathiae type 1a (the number of viable bacteria contained in the bacterial liquid is 2.6 multiplied by 10)5CFU), all piglets were observed daily for morbidity to day 14. The results are shown in Table 3.
TABLE 3 grouping results of piglets prepared from hyperimmune serum of Erysipelothrix rhusiopathiae
Note: "-" indicates inapplicable.
2) Serum separation and antibody titer detection
Before first immunity, after basic immunity, after first boosting immunity, after second boosting immunity, after first counteracting toxic substance and after second counteracting toxic substance, jugular vein blood sampling is carried out on each group of piglets and serum is separated.
Antibody levels in sera from immunized piglets were determined by the bacterial ELISA method, as shown in test example 2, and the results are shown in Table 4. And (3) after the antibody titer meets the requirement, filtering and sterilizing the serum by using a filter membrane of 0.22 mu m, and subpackaging to obtain the hyperimmune serum of the erysipelothrix rhusiopathiae.
TABLE 4 antibody levels in sera of immunized piglets at different immunization stages
Experiments prove that the pig erysipelas inactivated vaccine prepared by mixing the ginseng polysaccharide, the astragalus polysaccharide, the angelica polysaccharide and the epimedium polysaccharide (the adding ratio of each component is 2:1:1:1) with antigen liquid and matching with a good adjuvant can carry out multiple immunizations and virus attack on piglets, and the antibody level in the body of the piglets can be gradually improved, so that the hyperimmune serum with higher antibody titer can be prepared by the method.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the invention, so that any modifications, equivalents, improvements and the like, which are within the spirit and principle of the present invention, should be included in the scope of the present invention.
Claims (10)
1. The hyperimmune serum of the erysipelothrix rhusiopathiae is characterized in that: the swine erysipelas inactivated vaccine is prepared by taking a swine erysipelas mycelium 1a type strain as an antigen and matching with a traditional Chinese medicine immunopotentiator, wherein the traditional Chinese medicine immunopotentiator is one or more of ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide.
2. The erysipelothrix rhusiopathiae hyperimmune serum according to claim 1, characterized in that: the traditional Chinese medicine immunopotentiator is prepared from ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide, wherein the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide to the angelica polysaccharide to the epimedium polysaccharide is (2-3):1:1: 1; preferably, the mass ratio of the ginseng polysaccharide to the astragalus polysaccharide to the angelica polysaccharide to the epimedium polysaccharide is 2:1:1: 1; preferably, the mass ratio of the antigen to the traditional Chinese medicine immunopotentiator is 4: 1.
3. the erysipelothrix rhusiopathiae hyperimmune serum according to claim 1, characterized in that: the swine erysipelas inactivated vaccine also comprises an adjuvant, wherein the adjuvant is a mineral oil adjuvant or a Freund adjuvant; preferably, the swine erysipelas inactivated vaccine further comprises a preservative, and the preservative is thimerosal or phenol.
4. The erysipelothrix rhusiopathiae hyperimmune serum according to claim 1, characterized in that: in the swine erysipelas inactivated vaccine, the content of the inactivated thallus of the swine erysipelas 1a type strain is not less than 3.0 multiplied by 109CFU/mL。
5. A method for preparing the erysipelothrix rhusiopathiae hyperimmune serum according to any one of claims 1 to 4, comprising the steps of: the method comprises the following steps:
1) culturing erysipelothrix rhusiopathiae type 1a strain and inactivating the strain liquid;
2) concentrating the inactivated bacterial liquid, removing supernatant, washing and diluting to obtain antigen liquid;
3) mixing the antigen solution with traditional Chinese medicine immunopotentiator, adjuvant and antiseptic uniformly to obtain swine erysipelas inactivated vaccine;
4) carrying out 1 primary immunization on piglets by using the swine erysipelas inactivated vaccine, carrying out primary boosting immunization after 21d, carrying out secondary boosting immunization at an interval of 21d, carrying out primary challenge by using the swine erysipelas 1a type strain after 14d of immunization, and carrying out secondary challenge after 14d of interval;
5) collecting blood, separating serum, testing antibody titer to 210Bleeding all the carotid arteries of the piglets;
6) placing the collected blood in a constant temperature incubator at 37 ℃ for 1h, and then placing the blood in a refrigerator at 4 ℃ for 12-16 h; centrifuging the collected blood at 3000r/min for 5min, separating supernatant to obtain serum, filtering with 0.22 μm filter membrane, and packaging to obtain hyperimmune serum of Erysipelothrix rhusiopathiae;
7) the antibody level in the serum is detected by using a thallus ELISA method, and the titer reaches 210The hyperimmune serum of the erysipelothrix rhusiopathiae is obtained.
6. The method for preparing hyperimmune serum of Erysipelothrix rhusiopathiae according to claim 5, wherein the serum comprises the following steps: in the step 1), the content of viable bacteria of the erysipelothrix rhusiopathiae type 1a strain in the bacterial liquid is not less than 1.0 multiplied by 109CFU/mL。
7. The method for preparing hyperimmune serum of Erysipelothrix rhusiopathiae according to claim 5, wherein the serum comprises the following steps: in the step 1), a formaldehyde or beta-propiolactone inactivating agent is used for inactivation; preferably, the formaldehyde inactivating agent is used for inactivation, and is added into a formaldehyde solution with the mass concentration of 37-40% according to 0.25-0.3% of the total volume fraction of the bacterial liquid; inactivating at 37-39 deg.C for 24-48 h.
8. The method for preparing hyperimmune serum of Erysipelothrix rhusiopathiae according to claim 5, wherein the serum comprises the following steps: in the step 2), sterile PBS solution with the concentration of 0.0l mol/L, pH value of 7.2 is adopted for washing and diluting; after washing, the bacterial antigens were diluted with sterile PBS.
9. The method for preparing hyperimmune serum of Erysipelothrix rhusiopathiae according to claim 5, wherein the serum comprises the following steps: in the step 3), the traditional Chinese medicine immunopotentiator is obtained by mixing ginseng polysaccharide, astragalus polysaccharide, angelica polysaccharide and epimedium polysaccharide according to the mass ratio of (2-3) to 1:1:1, and the antigen liquid and the traditional Chinese medicine immunopotentiator are mixed according to the mass ratio of 4: 1; preferably, the adjuvant in the step 3) is a mineral oil adjuvant, the antigen liquid of the traditional Chinese medicine immunopotentiator and the mineral oil adjuvant are mixed according to the volume ratio of 1:2, and are emulsified for 5-10min at room temperature of 8000-10000r/min until the mixture is uniformly mixed; preferably, the adjuvant in the step 3) is Freund's adjuvant, and the antigen liquid of the traditional Chinese medicine immunopotentiator is mixed with Freund's adjuvantMixing the adjuvants at a volume ratio of 1: 1; emulsifying at room temperature 8000-; preferably, in the step 3), the preservative is thimerosal, and the addition amount of the preservative is 0.005% of the volume of the antigen liquid; preferably, in the step 3), the preservative is phenol, and the addition amount of the preservative is 0.3% of the volume of the antigen liquid; preferably, in the step 3), the content of the inactivated erysipelas suis type 1a strain in the inactivated erysipelas suis vaccine is not less than 3.0 x 109CFU/mL。
10. The method for preparing hyperimmune serum of Erysipelothrix rhusiopathiae according to claim 5, wherein the serum comprises the following steps: in the step 4), the immunization route is neck muscle immunization, and the dosage is 2 mL/head; preferably, in step 7), the antibody detection method is a thallus ELISA detection antibody, namely, the inactivated thallus is crushed to be clear, then the thallus is centrifuged at 12000r/min for 10min, cell fragments are discarded, and the supernatant is an antigen for ELISA detection.
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