CN114805554B - Refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and preparation method thereof - Google Patents
Refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a bivalent tetravalent refined egg yolk antibody injection which is colorless and transparent, has high titer and can be compared with human immunity protein and is used for resisting streptococcus suis and haemophilus parasuis. The yolk antibody contains antigens of haemophilus parasuis serotype 4, serotype 5, serotype 13 and streptococcus suis type 2; the haemophilus parasuis serotype 4 strain has a accession number of CVCC NO:3953; the haemophilus parasuis serotype 5 strain has a accession number of CVCC NO:3956; the haemophilus parasuis serum 13 type strain has a accession number of CVCC NO:3958; the preservation number of the streptococcus suis type 2 strain is CCTCC NO: m2021457. The purity of the yolk antibody can reach more than 90%, the maximum antibody titer can reach 8log2, and the antibody level can be maintained above 7log2 after the yolk antibody is stored for 1 year at normal temperature.
Description
Technical Field
The invention belongs to the field of preparation and application of egg yolk antibodies, and in particular relates to a refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and a preparation method thereof.
Background
Streptococcus Suis (SS) and haemophilus parasuis (Haemophilus parasuis, HPS) are two important pathogenic bacteria of pigs causing upper respiratory infectious diseases, and are widely and permanently existing in domestic pig groups, so that problems of slow growth, low immunity and the like of pig groups are caused, and healthy cultivation of live pigs is seriously affected.
Streptococcus Suis (SS) is gram positive, aerobic or facultative anaerobic bacteria, which not only invade the blood circulation to cause pneumonia, arthritis, lymphadenitis, etc., but also can cause meningitis and sow abortion through the blood brain barrier and placenta barrier. Meanwhile, streptococcus suis can infect people through wounds, respiratory tracts and other ways, and cause joint suppurative inflammation, meningoepithymitis, endocarditis and the like of the people, and serious death can be caused, which is listed as a national second-class animal epidemic disease. Streptococcus suis has a total of 33 serotypes, of which type 2 (SS 2) is the most prevalent and is a zoonotic. Haemophilus Parasuis (HPS) is a short-rod gram-negative bacterium that can cause swine grignard disease and is characterized by hyperthermia, joint swelling, airway disorders, and central nervous needles. Piglets under 10 days of age are very easy to infect from the sow with bacteria, and the weaned piglets often suffer from the decrease of the maternal antibody level. The haemophilus parasuis that has been identified at present includes 15 serotypes, most commonly of which are types 4 and 5. Different serotypes have different virulence, wherein the strains of type 4, type 5 and type 13 are medium and high virulence strains, often cause symptoms such as myocarditis, pneumonia, pleurisy, meningitis, arthritis and the like of pigs, and cause great threat to healthy breeding of live pigs.
Currently, the primary methods of preventing and treating streptococcus suis and haemophilus parasuis infections are vaccination and antibiotic therapy. In the aspect of prevention, inactivated vaccines are mainly adopted in China.
Patent CN101721696a discloses a trivalent oil emulsion inactivated vaccine of haemophilus parasuis serotype 4, type 5 and type 13. The preparation method is characterized in that three main epidemic serotypes and isolates of haemophilus parasuis in China are used as antigens, after the culture and propagation of TSB broth are used for 24 hours, the antigens of the three serotypes are inactivated by 0.3% formaldehyde for 11-13 hours, the mixture is mixed in the ratio of 1:1:1, then an oil phase which is equal to the total volume of the antigens is added, and the mixture is emulsified to prepare the haemophilus parasuis. The vaccine adjuvant is white oil, and the adjuvant vaccine has great side reaction and can form inflammation or swelling at the injection position; in addition, research data show that the use of mineral oil adjuvant can excite and improve the incidence of swine diseases such as porcine circus to a certain extent. In addition, in the preparation process of the vaccine, the obtained antigen cannot be used as an antigen for vaccine preparation because the obtained antigen is not judged and related tests of the inactivation effect after inactivation, and whether the antigen inactivation is complete cannot be known.
Along with the epidemic pathogenic strain and mixed infection, the existing vaccine is difficult to achieve the expected immune effect, the protection time of the antibody generated by the vaccine is short, the stronger immunity can be obtained only by multiple immunizations, and the stress on pigs is also large; there are also treatments with sensitive antibiotics, but with the massive application of antibiotics, resistance is becoming serious, the treatment effect is poor, and there are problems of antibiotic residues in meat products, etc.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis and a preparation method thereof, and the egg yolk antibody is colorless and transparent, has high titer and better safety compared with human immunity proteins.
The yolk antibody has no drug residue and drug resistance, is a safe, efficient and nuisanceless biological product, and can be used for daily prevention and treatment of streptococcus suis and haemophilus parasuis instead of antibiotics.
In order to solve the technical problems, the invention adopts the following technical scheme:
in a first aspect the present invention provides a yolk antibody against streptococcus suis and haemophilus parasuis comprising haemophilus parasuis antigens of serogroup 4, serogroup 5, serogroup 13 and streptococcus suis type 2;
the haemophilus parasuis serotype 4 strain has a accession number of CVCC NO:3953;
the haemophilus parasuis serotype 5 strain has a accession number of CVCC NO:3956;
the haemophilus parasuis serum 13 type strain has a accession number of CVCC NO:3958;
the preservation number of the streptococcus suis type 2 strain is CCTCC NO: m2021457.
The term "antigen" also known as an immunogen, refers to any substance that stimulates an immune response, is a molecule capable of being bound by antibodies, and is also capable of inducing a humoral and/or cellular immune response that produces B-and/or T-cells, and also has one or more epitopes (B-and T-epitopes). The antigen includes killed, inactivated bacteria, or a cultured cell preparation, supernatant thereof. Where the killed, inactivated bacteria is meant to be one that contains infectious organisms or pathogens that are no longer capable of replication or growth, the pathogen may be maintained in its immunogenicity by a variety of methods including freeze thawing, chemical treatment (e.g., treatment with thimerosal or formalin), sonication, irradiation, heat or any other conventional method sufficient to prevent replication or growth of the organisms.
Further, the bivalent quadrivalent inactivated vaccine contains haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 antigen diluent, and the colony number before inactivation is not less than 4 multiplied by 10 respectively 9 CFU/mL。
Further, the method comprises the steps of,
in the yolk antibody, inactivated antigen diluents of haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 are mixed in equal volumes.
Further, the total content of the inactivated antigen dilutions of haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 is 50% (V/V) of the total vaccine.
Further, the bivalent tetravalent inactivated vaccine also comprises an adjuvant; the adjuvant content was 50% (V/V) of the total vaccine.
The term "total vaccine amount" as used herein refers to the total amount of antigen and adjuvant.
The term "adjuvant" refers to a compound that, when administered in conjunction with an antigen, enhances the immune response of a subject to that antigen.
Further, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; further Freund's complete adjuvant.
In a second aspect, the present invention provides a method for preparing the egg yolk antibody as described above, comprising:
(1) Preparing bivalent tetravalent inactivated vaccine from haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 antigen;
(2) And immunizing the laying hens with the bivalent tetravalent inactivated vaccine and separating and extracting yolk antibodies in the immunized eggs.
The antibody prepared by the haemophilus parasuis serum 4, 5 and 13 strains can generate good immune protection effects on three serotypes, and if only haemophilus parasuis serum 4 and 5 are selected to prepare the inactivated vaccine, the immune protection effect on 13 strains is poor, even immune protection cannot be generated.
Further, the immunization regimen was 4 intramuscular injections into the leg and/or chest, each at 2 week intervals.
Further, when the antibody titer reaches 1:128, the immunized eggs are collected, and the egg yolk antibody is separated and extracted.
The third aspect of the invention provides an application of the egg yolk antibody in preparing a product for detecting, preventing and/or treating related diseases caused by haemophilus parasuis and streptococcus suis infection;
the product is selected from a formulation, a medicament or a feed additive.
The term "preventing and/or treating" when referring to a bacterial infection refers to inhibiting replication of the bacteria, inhibiting the spread of the bacteria or preventing colonization of the bacteria in its host, and alleviating the symptoms of the disease or disorder in which the bacterial infection is located. The treatment is considered to be therapeutic if the bacterial load is reduced, the symptoms of infection are reduced, and/or the food intake and/or growth is increased.
The beneficial effects are that:
the invention realizes the treatment and prevention of the haemophilus parasuis serum 4, 5, 13 and 2 type streptococcus suis for the first time, the egg yolk antibody can be used as feed additive or therapeutic biological agent, and the existing inactivated vaccine is only used for immunization.
The refined yolk antibody injection is colorless and transparent, has high titer, can be compared with human immunity protein, has the antibody purity of more than 90 percent, can reach the antibody titer of 8log2 at most, can maintain the antibody level of more than 7log2 after being stored for 1 year under normal temperature, has better safety, can achieve the effect of one needle with more prevention clinically, reduces the cost and can meet the requirements of different users.
Drawings
FIG. 1 shows the purification of IgY against Streptococcus suis and SDS-PAGE detection, and the purified IgY on the left; m is a protein Marker; 1.2 respectively represents the purified IgY protein bands against streptococcus suis and haemophilus parasuis.
Detailed Description
The present invention will be described in detail with reference to examples, which are illustrative only and are not limiting the scope of application of the present invention.
If no special description exists, the experimental methods are all conventional methods; the biological materials are all commercially available.
Example 1
1. Method of
1.1 test animals
BALB/c mice, 15-20 g, purchased from Sichuan Dada laboratory animal center; the 160-day-old Roman commodity generation laying hen, sichuan is big.
1.2 preservation of seed
Streptococcus suis type 2 virulent isolate SMU-2020-ZQ2 isolated from pigs dying of septicemia in pig farm in Mianyang city of Sichuan province, half the lethal mass (LD) of BALB/c mice 50 ) Is 2.1X10 7 CFU, strain is preserved in China center for type culture Collection, with the address being the eight-path 299 Wuhan university in Wuhan, hubei province, and the preservation number is CCTCC NO: M2021457. Latin name: streptomyces sp.
The haemophilus parasuis type 4, type 5 and type 13 virulent isolates are all from dead pigs suffering from respiratory symptoms in a pig farm in Leshan, sichuan province, and the strains are all preserved in China center for type-III veterinary microorganism strain preservation, and can be preserved in China center for type-III veterinary microorganism strain preservationThe heart was purchased with the address: guancun south street 8 in the sea lake area of Beijing city, the preservation numbers are CVCC NO:3953, CVCC NO: 3956. CVCC NO:3958, the preservation times are 25 days of 2008, 28 days of 2008, and LD for BALB/c mice, respectively 50 4.6X10 respectively 8 CFU，3.3×10 8 CFU and 5.6x10 8 CFU。
1.3 preparation of purified immune antigen
1.3.1 preparation of Streptococcus suis 2-type immune antigen
Streptococcus suis type 2 (SMU-2020-ZQ 2 strain) was inoculated onto tryptone soy agar medium (TSA) containing 5% calf serum, incubated in a constant temperature incubator at 37℃for 24h, single Colonies (CFU) were picked up and inoculated onto Tryptone Soy Broth (TSB) containing 5% calf serum for 18-24 h, diluted to 1X 10 with sterile PBS (0.01 mol, pH 7.2) 9 Adding formaldehyde into CFU/mL bacterial liquid to a final concentration of 0.3%, inactivating for 72 hours at 37 ℃, and shaking uniformly for 5-6 times during the inactivation; centrifuging at 6000rpm to separate precipitate thallus, re-suspending thallus with sterile PBS (0.01 mol, pH 7.2), centrifuging again, washing 2 times, diluting bacteria concentration with PBS to 4×10 9 CFU/mL bacterial liquid. 100 μl of the bacterial liquid was plated on a TSA culture plate of 5% bovine serum, and placed in a constant temperature incubator at 37deg.C for culturing for 72 hours, bacterial colonies were observed, and a negative control and an inactivated bacterial positive control were established simultaneously. If the positive control showed typical SS2 type colonies, but the medium inoculated with the inactivated bacterial liquid and the medium of the negative control group showed no colonies, the SMU-2020-ZQ2 bacterial liquid was completely inactivated.
1.3.2 preparation of type 4, type 5 and type 13 immune antigens from haemophilus parasuis
Haemophilus parasuis type 4 (3953 strain), type 5 (3956 strain) and type 13 (3958 strain) strains were inoculated onto TSA containing 5% calf serum and 1% Nicotinamide Adenine Dinucleotide (NAD), respectively, and cultured at 37℃for 24 to 36 hours. Picking single needle point size, smooth and transparent dew-shaped colony, inoculating again into TSB liquid culture medium containing 5% calf serum and 1% NAD, culturing for 24-36 hr, diluting to 1×10 with sterile PBS (0.01 mol, pH 7.2) 9 CFU/mL bacterial liquid, formaldehyde is added to a final concentration of 0.3%, and the bacterial liquid is inactivated at 37 ℃ for 72 hours, during which the bacterial liquid shakesShaking uniformly for 5-6 times; centrifuging at 6000rpm to separate precipitate thallus, re-suspending thallus with sterile PBS (0.01 mol, pH 7.2), centrifuging again, washing 2 times, diluting bacteria concentration with PBS to 4×10 9 CFU/mL bacterial liquid. 100 μl of the bacterial liquid was plated on TSA culture plates containing 5% calf serum and 1% NAD, cultured in a constant temperature incubator at 37deg.C for 72 hours, bacterial colonies were observed, and negative control and non-inactivated colony positive control were established simultaneously. If the positive control shows dew-shaped colonies with typical needle point size and smoothness and transparency, the medium inoculated with the inactivated bacterial liquid and the medium of the negative control group have no colonies, which indicates that the bacterial liquid is completely inactivated.
1.3.3 bacterin configuration
And (3) mixing the three serotype strains (3953 strain, 3956 strain and 3958 strain) of the streptococcus suis (SMU-2020-ZQ 2 strain) and haemophilus parasuis which are completely inactivated through detection in equal volumes, and mixing the mixture with Freund's complete adjuvant in equal volumes to form slightly viscous milky suspension, namely the streptococcus suis and haemophilus parasuis bivalent tetravalent inactivated vaccine respectively.
1.3.4 Security check
The safety test experiment steps of the inactivated vaccine are as follows:
1) And (3) sterile detection: 100 μl of the inactivated vaccine was spread on a TSA culture plate of 5% bovine serum, and after culturing at 37deg.C for 24 hours, the presence or absence of bacterial growth was observed.
2) And (3) safety detection: the inactivated vaccine is warmed at room temperature, 6-8 weeks old SPF grade BALB/c female mice are inoculated by subcutaneous multipoint injection at the neck and back, the inoculation dosage is 1 mL/mouse, the vaccine is fed into animal houses of medical laboratory of national university in southwest after inoculation, and abnormal conditions such as feeding, mental state, red swelling at injection position and the like are observed.
Safety inspection results: the inactivated vaccine is inoculated to a BALB/c mouse, so that the vaccine is full in spirit, normal in appetite, good in absorption at an injection site, free of any adverse and abnormal phenomena such as red swelling and the like, and the streptococcus suis and haemophilus parasuis bivalent and tetravalent inactivated vaccine prepared by the research has good safety and can be used for subsequent experiments.
1.4 immunization of chicken flocks and egg Collection
A high-yield Roman commodity layer chicken without main infectious diseases, especially salmonella pullorum, is selected, and is respectively injected with 1mL of streptococcus suis and haemophilus parasuis bivalent tetravalent inactivated vaccine prepared by the research through leg and/or chest muscle immunization, and is continuously immunized for 4 times at intervals of 2 weeks.
Serum assay titers were collected after immunization 4. When the antibody agglutination titer reached 1:2 7 The immunization is considered to be qualified, eggs are collected every day, marked and stored at 4 ℃ for standby.
1.5 determination of the titers of the bivalent tetravalent egg yolk antibodies against Streptococcus suis and Haemophilus parasuis
Randomly collecting 3 eggs every 7d of immune group and non-immune group after the 4 th immunization, sterilizing in 0.1% benzalkonium bromide aqueous solution at 42 ℃ for 15min, taking out, air drying, and separating yolk with an egg white yolk separator; putting yolk into a sterile beaker, blowing and mixing uniformly, taking 1mL of the uniformly mixed yolk, adding into 9mL of deionized water, mixing uniformly again, and adjusting the pH value of the yolk solution to 5.0-5.2; placing in a refrigerator at 4deg.C, standing for 4 hr, collecting supernatant, centrifuging at 6000rpm/min at 4deg.C for 25min, collecting supernatant, and storing.
Antibody titers were determined using a plate agglutination assay. 50 mu L of sterilized PBS (0.01 mol, pH 7.2) is added in the center of each grid of a glass plate, then 50 mu L of the treated yolk antibody sample is sucked into the first grid, after being fully and uniformly mixed by a pipetting gun head, 50 mu L of the mixed solution is sucked into the second grid, and the mixed solution is fully and uniformly mixed, and is diluted to the 8 th grid (namely, diluted 1024 times) by a multiple ratio in sequence, and 50 mu L of the mixed solution is sucked out and discarded. Panels 9, 10 and 11 served as antigen control and positive and negative serum control, respectively. Then 50. Mu.L of the antigen for detection was added to each cell of the droplets, and stirred with a toothpick. After 2 to 5 minutes, the agglutination reaction phenomenon of each glass plate cell is observed, the negative serum control cell is slightly even and turbid (the antigen is diluted by 2 times), the negative is judged as negative, the positive control cell is marked as "-", the positive control cell is marked as floccule, and the positive control cell is judged as positive, and the positive control cell is marked as "+". The more clots that appear, the more transparent the liquid, the stronger the positive response, the weak to strong are marked as "+", respectively "+," +++ "," # ". The highest serum dilution above "++" was the titer of this serum (egg yolk antibody had been 2log2 diluted during extraction).
The specific antibody is detected 7 days after the egg yolk antibody of the immunized chicken is subjected to secondary immunization, the antibody titer reaches 8log2, 9log2, 8log2 and 8log2 respectively after the streptococcus suis type 2, haemophilus parasuis type 4, type 5 and type 13 of 7d after the 4 th immunization, and the antibody level can be maintained above 7log2 after one year of the 4-time immunization.
1.6 extraction and purification of IgY antibodies
Collecting immunized qualified egg, sterilizing in 0.1% benzalkonium bromide water solution at 42deg.C for 15min, taking out, air drying, separating yolk with egg white and yolk separator, and weighing. Adding purified water according to the proportion of 1:6-9 to dilute and homogenate. Adjusting pH to 4.8-6.0,2-8deg.C with 1moL/L hydrochloric acid solution, standing for 1-24h. Centrifuging at 4000-20000 r/min, removing sediment, and taking supernatant. Adding 10% -35% ammonium sulfate into supernatant, salting out, mixing well, and precipitating overnight. And (3) obtaining a sediment 4000-20000 r/min, centrifugally desalting, obtaining a crude extract of the egg yolk antibody, and removing the supernatant of the ammonium sulfate. The obtained precipitate is re-dissolved by 1/150 to 1/300 volume of weak acid buffer solution, the pH value is adjusted to 4.8 to 6.0, the precipitate is fully dissolved at the temperature of 2 to 8 ℃ overnight, and the supernatant is taken. The supernatant is prepared and is subjected to 54-60 ℃ and the microorganism in the antibody is inactivated for 8-20 hours. The inactivated supernatant is ultrafiltered with ultrafiltering membrane of cut-off relative molecular weight of 50-100 kD. The ultrafiltrate is sterilized and filtered to obtain IgY stock solution. The IgY stock solution is prepared into a yolk antibody preparation by adding a stabilizer according to the detected antibody titer.
In the process of preliminary preparation of the egg yolk antibody, the purity of the crude egg yolk antibody is not high, and the protection efficiency shown in animal experiments is not improved, so that IgY can be further purified by ultrafiltration after being extracted by an ammonium sulfate method, the impurity proteins are reduced, the purity of the purified IgY is higher, the impurity bands are less, SDS-PAGE (sodium dodecyl sulfate-PAGE) non-reducing electrophoresis shows that the molecular weight of the extracted IgY against streptococcus suis and haemophilus parasuis is about 180kD, and the purity of the IgY antibody is more than 90% after gel imaging software analysis.
SDS-PAGE denaturing electrophoresis shows that the extracted anti-Streptococcus suis and haemophilus parasuis bigeminal tetravalent yolk antibody IgY is divided into two major bands, 65kDa being IgY heavy chain and 25kDa being IgY light chain. The determination shows that the agglutination titer of the purified IgY finished product on streptococcus suis type 2, haemophilus parasuis type 4, type 5 and type 13 can reach above 8log 2.
1.7 egg yolk IgY antibody hemolysis assay
Several milliliters of rabbit blood was collected aseptically, and the blood was stirred with a glass rod to remove fibrinogen and to form defibrinated blood. Adding 10 times of 0.9% sodium chloride solution, shaking, centrifuging at 1000-1500r/min for 15min, removing supernatant, and washing the precipitated red blood cells with 0.9% sodium chloride solution for 2-3 times.
The resulting erythrocytes were made into a 2% suspension with 0.9% sodium chloride solution. The prepared egg yolk antibody is diluted with 0.9% sodium chloride solution according to the ratio of 1:3 to be used as a test solution, sterile physiological saline is used as a negative control, and non-inactivated streptococcus suis cultures are used as a positive control, and are respectively added into an EP tube. Then, 2% erythrocyte suspension and 0.9% sodium chloride solution were added in this order, and after mixing, the mixture was immediately incubated in an incubator at 37.+ -. 0.5 ℃ for 1 time at 12-hour intervals, followed by 6 days of continuous observation, and the analysis result was recorded.
The results showed that no hemolysis and aggregation occurred in the negative control tube, and that hemolysis occurred in the positive control tube; meanwhile, the solution in the test tube does not hemolyze or agglomerate within 144 hours, which indicates that the yolk IgY antibody prepared by the research has no hemolysis and can be used by injection.
1.8 in vitro bacteriostasis test of Streptococcus suis and Haemophilus parasuis bivalent and tetravalent refined egg yolk antibody injection
1.8.1 evaluation of in vitro bacteriostatic Effect of Streptococcus suis 2 by means of a Streptococcus suis bivalent and tetravalent refined egg yolk antibody injection
Streptococcus suis SMU-2020-ZQ2 was inoculated into TSB containing 5% bovine serum and cultured to a bacterial concentration of 10 7 CFU/mL, 200 μl of diluted bacterial suspension and 200 μl of egg yolk IgY antibody or PBS (control).
After incubation for 1h and 2h at 37℃respectively, 10-fold dilution with PBS was performed, 0.1mL was plated on TSA medium containing 5% serum, and the bacterial colonies were counted for each well after incubation for 24h at 37℃respectively. Three replicates were made for each dilution, three replicates were made for each sample, 3 replicates each time. Percentage of bacterial reduction: [ CFU (bacterium+PBS) -CFU (bacterium+antibody) ]/CFU (bacterium+PBS). Times.100.
1.8.2 evaluation of in vitro bacteriostatic Effect of Streptococcus suis and Haemophilus Parasuis (HPS) bivalent tetravalent refined egg yolk antibody injection on three Haemophilus parasuis
Three different serotypes of haemophilus parasuis were inoculated into TSB containing 5% bovine serum and 1% NAD and cultured to a bacterial concentration of 10 7 CFU/mL。
After incubation of 200. Mu.L of diluted bacterial suspension and 200. Mu.L of yolk IgY antibody or PBS (control group) at 37℃for 1h and 2h, respectively, 10-fold dilution with PBS was performed, 0.1mL was plated on TSA medium containing 5% serum, and each well was incubated at 37℃for 24h, respectively, and the bacterial colony count was counted. Three replicates were made for each dilution, three replicates were made for each sample, 3 replicates each time. The percent bacterial reduction was calculated.
The results of the bacteriostasis experiments are shown in Table 1.
TABLE 1 bacterial content (cfu/ml) in culture broth at the same time of culture (h)
Compared with PBS group, igY finished products can obviously inhibit the growth of streptococcus suis and haemophilus parasuis in vitro.
1.9 Passive protection test of Streptococcus suis and Haemophilus parasuis bivalent and tetravalent refined egg yolk antibody injection against mice
80 BALB/c mice were randomly divided into 8 groups of 10 mice each, wherein 7 groups were treated with 1 drop of yolk IgY antibody 0.5mL per day, 3d was used in combination with 1 drop of physiological saline 0.5mL as a control group, and 5LD50 Streptococcus suis type II, haemophilus parasuis type 4, type 5 and type 13 mice were treated with each group after day 4, and the protection rate of the yolk IgY antibody on mice was judged by intramuscular injection of the challenged mice.
The results of passive protection test of the yolk IgY antibodies on mice are shown in table 2.
TABLE 2 results of passive protection test of IgY antibody to BALB/c mice
The results show that: mice with normal saline drops all show obvious clinical symptoms after toxin expelling: emaciation, dyspnea, depression of the quilt Mao Zaluan, appetite reduction and the like, death begins to occur 16 hours after toxin attack, and all death occurs within 48 hours. The mice with yolk IgY antibody drops have good protection effects on streptococcus suis type II, haemophilus parasuis type 4, type 5 and type 13 strains, and the protection efficiency is higher than 90%. 3d after the virus-fighting streptococcus suis II strain, 2 mice die, and the survival protection rate of the rest total mice is 90%; after the strain of haemophilus parasuis type 4 is attacked, 1 mouse is killed in 2d, and the survival protection rate of the rest whole mice is 90%; no mice die after the strain of haemophilus parasuis type 5 is attacked, and the protection rate is 100%; after the strain of haemophilus parasuis 13 is attacked, 1 mouse dies in 3d, and the survival protection rate of the rest whole mice is 90%.
In conclusion, the purity of IgY in the purified and refined egg yolk antibody of the bivalent and tetravalent refined egg yolk antibody injection for resisting streptococcus suis type 2 and haemophilus parasuis type 4, 5 and 13 provided by the invention can reach more than 90%, the IgY is not hemolyzed, and the agglutination titer of the bivalent and tetravalent refined egg yolk antibody for resisting streptococcus suis type 2, haemophilus parasuis type 4, 5 and 13 can reach more than 8log2; the obtained IgY finished product can obviously inhibit the growth of streptococcus suis type 2 and haemophilus parasuis type 4, type 5 and type 13 in vitro, and the protection rate of the virus-fighting mice is higher than 90%, especially the protection rate of haemophilus parasuis type 5 strain reaches 100%. Provides a safe, high-efficiency and nuisanceless biological product for clinical treatment of streptococcus suis and haemophilus parasuis, and can replace antibiotics to be used for daily prevention and treatment of streptococcus suis and auxiliary haemophilus parasuis.
The present invention is not limited to the above-mentioned embodiments, but is capable of other and obvious modifications and equivalents of the above-mentioned embodiments without departing from the scope of the present invention.
Claims (4)
1. A method for preparing a refined egg yolk antibody injection for resisting streptococcus suis and haemophilus parasuis, which is characterized by comprising the following steps:
(1) Preparing bivalent tetravalent inactivated vaccine from haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 antigen;
the haemophilus parasuis serotype 4 strain has a accession number of CVCC NO:3953;
the haemophilus parasuis serotype 5 strain has a accession number of CVCC NO:3956;
the haemophilus parasuis serum 13 type strain has a accession number of CVCC NO:3958;
the preservation number of the streptococcus suis type 2 strain is CCTCC NO: m2021457;
(2) Immunizing a laying hen with a bivalent tetravalent inactivated vaccine and separating and extracting yolk antibodies in the immunized eggs; the immunization mode is that intramuscular injection is performed on legs and/or chest for 4 times, and each time of the intramuscular injection is divided into 2 weeks;
when the antibody titer reaches 1:128, collecting immunized eggs 7d after the 4 th immunization, separating and extracting yolk antibody, wherein the antibody titers of streptococcus suis type 2, haemophilus parasuis type 4, type 5 and type 13 reach 8log2, 9log2, 8log2 and 8log2 respectively;
the bivalent quadrivalent inactivated vaccine contains haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 antigen diluent, and the colony number before inactivation is not less than 4×10 respectively 9 CFU /mL；
In the yolk antibody, inactivated antigen diluents of haemophilus parasuis serum 4 type, serum 5 type, serum 13 type and streptococcus suis type 2 are mixed in equal volumes.
2. The method for preparing the yolk antibody injection according to claim 1, wherein the total content of inactivated antigen diluents of haemophilus parasuis serotype 4, serotype 5, serotype 13 and streptococcus suis type 2 is 50% of the total vaccine
（V/V）。
3. The method for preparing the egg yolk antibody injection according to claim 1, wherein the bivalent tetravalent inactivated vaccine further comprises an adjuvant; the adjuvant content is 50% (V/V) of the total vaccine; the adjuvant is Freund's complete adjuvant.
4. Use of the egg yolk antibody of any one of claims 1-3 in the preparation of a product for detecting, preventing and/or treating related diseases caused by haemophilus parasuis serotype 4, serotype 5, serotype 13, streptococcus suis type 2 infection;
the product is selected from a formulation, a medicament or a feed additive.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721696A (en) * | 2009-12-18 | 2010-06-09 | 西南民族大学 | Trivalent oil emulsion inactivated vaccine for 4 type, 5 type and 13 type serum of haemophilus parasuis |
| CN102329746A (en) * | 2011-08-16 | 2012-01-25 | 武汉科前动物生物制品有限责任公司 | Porcine streptococcus disease and haemophilus parasuis disease combined inactivate vaccine and preparation method thereof |
| CN102716485A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Dual hyper-immune egg yolk antibody injection for duck virus hepatitis and duck plague and preparation method thereof |
| CN103157100A (en) * | 2011-12-08 | 2013-06-19 | 普莱柯生物工程股份有限公司 | hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof |
| CN103157101A (en) * | 2011-12-08 | 2013-06-19 | 普莱柯生物工程股份有限公司 | Combined inactivate vaccine for haemophilus parasuis disease and streptococcus suis disease and preparation method for same |
| US10279031B2 (en) * | 2016-05-11 | 2019-05-07 | Phibro Animal Health Corporation | Composition comprising antigens and a mucosal adjuvant and a method for using |
| AU2020103896A4 (en) * | 2020-12-04 | 2021-02-11 | Institute Of Animal Science And Veterinary Medicine, Shandong Academy Of Agricultural Sciences | A detection kit and method for haemophilus parasuis |
| CN114181846A (en) * | 2021-09-11 | 2022-03-15 | 江苏南农高科技股份有限公司 | Streptococcus suis and haemophilus parasuis combined inactivated vaccine and preparation method thereof |
| CN114377127A (en) * | 2022-01-25 | 2022-04-22 | 天津市中升挑战生物科技有限公司 | Triple egg yolk antibody preparation and preparation method and application thereof |
| CN114634564A (en) * | 2022-04-18 | 2022-06-17 | 北京华驰千盛生物科技有限公司 | Triple egg yolk antibody for cat, preparation method and application |
| CN115887631A (en) * | 2022-12-23 | 2023-04-04 | 山东信得科技股份有限公司 | Haemophilus parasuis and streptococcus suis combined vaccine |
-
2022
- 2022-04-13 CN CN202210389078.1A patent/CN114805554B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101721696A (en) * | 2009-12-18 | 2010-06-09 | 西南民族大学 | Trivalent oil emulsion inactivated vaccine for 4 type, 5 type and 13 type serum of haemophilus parasuis |
| CN102329746A (en) * | 2011-08-16 | 2012-01-25 | 武汉科前动物生物制品有限责任公司 | Porcine streptococcus disease and haemophilus parasuis disease combined inactivate vaccine and preparation method thereof |
| CN103157100A (en) * | 2011-12-08 | 2013-06-19 | 普莱柯生物工程股份有限公司 | hemophilus parasuis disease, swine streptococcosis bivalent inactivated vaccine and preparation method thereof |
| CN103157101A (en) * | 2011-12-08 | 2013-06-19 | 普莱柯生物工程股份有限公司 | Combined inactivate vaccine for haemophilus parasuis disease and streptococcus suis disease and preparation method for same |
| CN102716485A (en) * | 2012-05-31 | 2012-10-10 | 郑州后羿制药有限公司 | Dual hyper-immune egg yolk antibody injection for duck virus hepatitis and duck plague and preparation method thereof |
| US10279031B2 (en) * | 2016-05-11 | 2019-05-07 | Phibro Animal Health Corporation | Composition comprising antigens and a mucosal adjuvant and a method for using |
| AU2020103896A4 (en) * | 2020-12-04 | 2021-02-11 | Institute Of Animal Science And Veterinary Medicine, Shandong Academy Of Agricultural Sciences | A detection kit and method for haemophilus parasuis |
| CN114181846A (en) * | 2021-09-11 | 2022-03-15 | 江苏南农高科技股份有限公司 | Streptococcus suis and haemophilus parasuis combined inactivated vaccine and preparation method thereof |
| CN114377127A (en) * | 2022-01-25 | 2022-04-22 | 天津市中升挑战生物科技有限公司 | Triple egg yolk antibody preparation and preparation method and application thereof |
| CN114634564A (en) * | 2022-04-18 | 2022-06-17 | 北京华驰千盛生物科技有限公司 | Triple egg yolk antibody for cat, preparation method and application |
| CN115887631A (en) * | 2022-12-23 | 2023-04-04 | 山东信得科技股份有限公司 | Haemophilus parasuis and streptococcus suis combined vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 鸡法氏囊和腺病毒精制卵黄抗体制备与应用;吴卫东;中国畜禽种业;第15卷(第5期);185-186 * |
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