Preparation method of a Riemerella anatipestifer complex vaccine and yolk antibody

Abstract

The present application discloses a preparation method of a Riemerella anatipestifer composite vaccine and yolk antibody, which belongs to the field of biotechnology. The Riemerella anatipestifer capsular polysaccharide is mixed with the recombinant PVAX1-OmpA plasmid to prepare the Riemerella anatipestifer composite vaccine; after immunizing laying hens with the composite vaccine, the egg collection requirement can be met 7 days after the third immunization. In terms of the yolk antibody production time, antibody production level and antibody persistence period, it is significantly superior to the whole cell inactivated vaccine; and when detected 2 months after the third immunization, the yolk antibody titer still meets the requirements. The yolk antibody has a good clinical protection effect on ducklings infected with Riemerella anatipestifer and can be used for the prevention and treatment of Riemerella anatipestifer; this method can also reduce the preparation cost of the Riemerella anatipestifer yolk antibody, reduce the immune stress of laying hens, and has great popularization and application value.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and particularly relates to a method for preparing a combined vaccine against Riemerella anatipestifer and yolk antibody. Background Art

[0002] Riemerella anatipestifer is a Gram-negative rod-shaped bacterium without flagella, spores, and can form a capsule in the genus Riemerella of the family Flavobacteriaceae. It is a facultative anaerobe and can grow on tryptone soy agar medium, blood agar medium, and chocolate agar medium. Riemerella anatipestifer infects multiple breeds of ducks such as Cherry Valley ducks, Shelducks, and Muscovy ducks, and ducks aged 1 - 5 weeks are the most susceptible. The clinical symptoms of diseased ducks first show listlessness, reduced food intake, tearing, sneezing, and greenish loose stools, and then ataxia and head and neck tremors occur. In the later stage, the number of ducks with neurological symptoms increases significantly. After autopsy of diseased ducks, it is mainly manifested that there is fibrinoid exudation on the serosal surface of organs, which is most obvious in the heart and liver. The incidence and mortality rates range from 5% to 75%, and the surviving ducks that have survived the infection grow slowly, and their body weight is significantly less than that of normal ducks. Some ducks have sequelae of encephalitis and become "stiff ducks" or "disabled ducks". Riemerella anatipestifer has a wide range of prevalence, high infection and mortality rates, causing huge economic losses to the duck farming industry and being one of the most common diseases endangering duck breeding.

[0003] Due to the serious drug resistance of Riemerella anatipestifer, the number of failed cases in clinical treatment with antibiotics has increased year by year. At the same time, the treatment of Riemerella anatipestifer with antibiotics will cause a series of problems such as food safety and environmental pollution. Therefore, how to develop more effective products for reducing or replacing antibiotics for the prevention and control of Riemerella anatipestifer is a current research hotspot. Yolk antibody is a type of specific IgY antibody extracted from hyperimmune eggs produced after immunizing laying hens, and is commonly used for the prevention and control of clinical viral and bacterial diseases, with the characteristics of low price, environmental friendliness, strong specificity, and rapid onset. Riemerella anatipestifer is a Gram-negative bacterium, and its cells contain a large amount of bacterial endotoxin. Directly making a whole-cell inactivated vaccine and immunizing laying hens with multiple doses is extremely likely to damage the oviduct of laying hens and reduce egg production, resulting in a lag in the development of yolk antibody against Riemerella anatipestifer. Summary of the Invention

[0004] The purpose of the implementation of this application is to provide a method for preparing a combined vaccine against Riemerella anatipestifer and yolk antibody to solve the technical problems of excessive side effects, poor effect, and short duration of the whole-cell inactivated vaccine in the prior art.

[0005] To achieve the above purpose, the technical solution adopted in this application is: providing a method for preparing a combined vaccine against Riemerella anatipestifer, including the following steps:

[0006] (1). Mix the capsular polysaccharide solution and the recombinant PVAX1 - OmpA plasmid in proportion, and inactivate to obtain a semi-finished product;

[0007] (2) Mix white oil for injection and aluminum stearate, add Span 80 after heating, raise the temperature and mix to obtain an oil phase; mix Tween 80 and the semi-finished product to obtain an aqueous phase; mix the oil phase and the aqueous phase for emulsification to obtain a complex vaccine against Riemerella anatipestifer.

[0008] In one embodiment,

[0009] The preparation method of the capsular polysaccharide solution is as follows: ferment and culture Riemerella anatipestifer to obtain a fermentation broth, centrifuge the fermentation broth to take the supernatant, add cetyltrimethylammonium bromide solution and then centrifuge to discard the supernatant, resuspend the precipitate with sodium chloride solution, add ethanol and then centrifuge to discard the supernatant, dissolve the precipitate to obtain the capsular polysaccharide solution.

[0010] In one embodiment,

[0011] The final concentration of the Riemerella anatipestifer fermentation broth after adding the cetyltrimethylammonium bromide solution is 5 mM; the concentration of the sodium chloride solution is 0.4 M, and the concentration of the ethanol is 95%; the precipitate is dissolved with physiological saline; the inactivation is carried out with a formaldehyde solution.

[0012] In one embodiment,

[0013] The preparation method of the recombinant PVAX1-OmpA plasmid is as follows:

[0014] (1) Construction of the vector of the recombinant PVAX1-OmpA plasmid: The outer membrane protein OmpA gene of Riemerella anatipestifer is synthesized by total gene synthesis, and the outer membrane protein OmpA gene is ligated with the eukaryotic expression vector PVAX1 by restriction enzyme digestion to construct a recombinant PVAX1-OmpA plasmid, and it is transferred into Escherichia coli DH5α;

[0015] (2) Large-scale preparation of the recombinant PVAX1-OmpA plasmid: After fermenting and culturing the Escherichia coli DH5α, centrifuge to collect the bacterial cells, successively add hydrochloric acid buffer solution, sodium hydroxide solution and acetic acid solution, and carry out post-treatment to obtain a purified recombinant PVAX1-OmpA plasmid solution; remove the bacterial endotoxin in the purified recombinant PVAX1-OmpA plasmid solution to obtain a large-scale prepared recombinant PVAX1-OmpA plasmid.

[0016] In one embodiment,

[0017] In step (2), the ratio of the bacterial cells to the hydrochloric acid buffer solution is 1 g: 5 ml, and the volume ratio of the hydrochloric acid buffer solution, sodium hydroxide solution and acetic acid solution is 1: 2: 1.5.

[0018] In one embodiment,

[0019] The formulation of the hydrochloric acid buffer solution is 25 mM Tris-HCl, 10 mM EDTA, and 50 mM glucose; the formulation of the sodium hydroxide solution is 250 mM NaOH and 1% SDS; the formulation of the acetic acid solution is 3 M potassium acetate and 5 M acetic acid.

[0020] In one embodiment,

[0021] The final concentration of capsular polysaccharide in the capsular polysaccharide solution is 0.1 - 0.6 mg / ml, and the final concentration of the recombinant PVAX1-OmpA plasmid is 10 - 100 µg / ml. Preferably, the final concentration of capsular polysaccharide is 0.3 mg / ml, and the final concentration of the recombinant PVAX1-OmpA plasmid is 50 µg / ml.

[0022] In one embodiment,

[0023] By weight, white oil for injection is 94 parts, aluminum stearate is 2 parts, Span 80 is 6 parts, Tween 80 is 4 parts, semi-finished product is 96 parts, oil phase is 2 parts, and water phase is 1 part.

[0024] The present application also provides a method for preparing Riemerella anatipestifer yolk antibody, which specifically includes the following steps: using the composite vaccine prepared by the preparation method of the Riemerella anatipestifer composite vaccine in any of the above embodiments to immunize laying hens, collecting eggs after immunization to obtain hyperimmune eggs, separating egg yolk, extracting with octanoic acid, inactivating, filtering and sterilizing, and packaging the hyperimmune eggs to obtain Riemerella anatipestifer yolk antibody.

[0025] In one embodiment,

[0026] The number of times of vaccine immunization is three, with an interval of 14 days each time, and the injection volume of the vaccine is 0.5 ml / hen.

[0027] The present application provides a preparation method of a Riemerella anatipestifer composite vaccine and yolk antibody. After immunizing laying hens with the composite vaccine, the requirement for collecting eggs can be met 7 days after the third immunization. In terms of the yolk antibody production time, antibody production level, and antibody persistence period, it is significantly superior to the whole-cell inactivated vaccine; and when detected 2 months after the third immunization, the titer of the yolk antibody still meets the requirements. The yolk antibody has a good clinical protection effect on ducklings infected with Riemerella anatipestifer and can be used for the prevention and treatment of Riemerella anatipestifer; this method can also reduce the preparation cost of Riemerella anatipestifer yolk antibody and reduce the immune stress of laying hens, and has great popularization and application value. Detailed implementation mode

[0028] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer and more understandable, this application is further described in detail. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0029] Riemerella anatipestifer was obtained commercially.

[0030] Example 1

[0031] (I). Preparation of capsular polysaccharide solution

[0032] 1. Preparation of production seeds

[0033] Streak inoculate the Riemerella anatipestifer seeds on a chocolate nutrient agar plate and culture them in a candle jar at 37 °C for 16 - 24 h. Select 10 typical colonies and inoculate them into 100 ml of LB culture medium, and culture them at 37 °C for 8 - 10 h to obtain a seed solution.

[0034] 2. Preparation of culture medium

[0035] The fermentation medium is tryptone soy broth medium. Each 1000 ml of the medium contains 17 g of tryptone, 3 g of soy peptone, 5 g of sodium chloride, 2.5 g of glucose, and 2.5 g of dipotassium hydrogen phosphate.

[0036] 3. Bacterial liquid culture

[0037] Load an appropriate amount of culture medium (accounting for 60 - 80% of the culture tank volume) and antifoaming agent into the culture tank. After sterilization, inoculate the seed solution at 1% of the culture medium amount, adjust the rotation speed and pH value to keep the dissolved oxygen above 60% and the pH value around 7.2, and culture it aerobically at 37 °C for 6 - 8 h to obtain a fermentation broth.

[0038] 4. Preparation of Riemerella anatipestifer capsular polysaccharide solution

[0039] S1. Centrifuge the fermentation broth at 8000 r / min for 10 min, discard the bacterial cell precipitate. Add a CTAB solution with a final concentration of 5 mmol / L to the supernatant, mix well, then refrigerate and stand for 2 h. Then centrifuge at 8000 r / min for 10 min, discard the supernatant, dissolve the precipitate with a small amount of 0.4 mol / l sodium chloride solution, add 5 volumes of 95% ethanol, mix well, and stand overnight at 4 °C. Centrifuge at 8000 r / min for 10 min, discard the supernatant, and dissolve the precipitate with sterile normal saline to obtain the Riemerella anatipestifer capsular polysaccharide solution.

[0040] S2. Use the anthrone method to determine the content of Riemerella anatipestifer capsular polysaccharide, and the content of capsular polysaccharide should not be less than 1 mg / ml.

[0041] (2) Preparation of recombinant PVAX1-OmpA plasmid

[0042] 1. Construction of the vector of recombinant PVAX1-OmpA plasmid

[0043] S1: According to the OmpA gene sequence of Riemerella anatipestifer strain JL-RA3 on NCBI (GenBank Number: HQ707078), double restriction enzyme sites of Kpn I and Xho I were added to both ends of the OmpA gene, and then the whole gene was synthesized to obtain the synthesized outer membrane protein OmpA gene;

[0044] S2: The outer membrane protein OmpA gene was ligated into the corresponding restriction enzyme site of the eukaryotic expression vector PVAX1. The plasmid was extracted and identified by double digestion with Kpn I and Xho I. After being correct, sequencing was carried out (the sequencing results showed that the OmpA gene of Riemerella anatipestifer was successfully ligated into the corresponding restriction enzyme site of the PVAX1 vector), and the recombinant PVAX1-OmpA plasmid was successfully constructed; It was transformed into Escherichia coli DH5α by the CaCl2 method, and the recombinant PVAX1-OmpA plasmid was transferred into Escherichia coli DH5α;

[0045] 2. Large-scale preparation of recombinant PVAX1-OmpA plasmid

[0046] S1: High-density fermentation and cell lysis of the recombinant bacteria

[0047] After the Escherichia coli DH5α containing the recombinant PVAX1-OmpA plasmid DNA was cultured by high-density fermentation in a 50 L fermenter, the cells were collected by centrifugation at 5000 r / min for 10 min. Hydrochloric acid buffer was added according to the ratio of 5 ml per 1 g of wet bacteria, and then hydrochloric acid buffer, sodium hydroxide solution and acetic acid solution were added according to the volume ratio of 1:2:1.5. After incubation at room temperature for 15 min, centrifugation was carried out at 10000 r / min at room temperature for 10 min, and the supernatant was collected. 0.7 times the volume of isopropanol was added, and precipitation was carried out at -20 °C for 30 min. Centrifugation was carried out at 10000 r / min at room temperature for 10 min, the supernatant was discarded, and the precipitate was dissolved in 10 mmol / L TE buffer to obtain a purified plasmid DNA solution;

[0048] The formula of the hydrochloric acid buffer is 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose;

[0049] The formula of the sodium hydroxide solution is 250 mM NaOH, 1% (w / v) SDS;

[0050] The formula of the acetic acid solution is 3 M potassium acetate, 5 M acetic acid;

[0051] S2: Removal of bacterial endotoxin

[0052] Add the purified plasmid solution to 10% Triton X-114 to make the final concentration 1%. After mixing evenly, incubate on ice for 10 min, continue to incubate at 42 °C for 10 min, centrifuge at 10000 r / min at room temperature for 10 min, carefully aspirate the supernatant into a pyrogen-free container, and repeat the operation once if necessary; dilute with 10 mmol / L TE buffer to 500 µg / ml, quantitatively dispense, and obtain the recombinant PVAX1-OmpA plasmid;

[0053] (III) Preparation of Riemerella anatipestifer complex vaccine

[0054] 1. Preparation of semi-finished product

[0055] Mix the Riemerella anatipestifer capsular polysaccharide solution prepared in step (I) with the diluted recombinant PVAX1-OmpA plasmid prepared in step (II) so that the final concentration of capsular polysaccharide is 0.3 mg / ml and the final concentration of DNA in the recombinant PVAX1-OmpA plasmid is 50 µg / ml. Add formaldehyde with a final concentration of 0.2% and inactivate at 37 °C for 24 h to obtain the semi-finished product;

[0056] 2. Vaccine preparation

[0057] S1. Preparation of oil phase

[0058] Take 94 parts of high-quality white oil for injection and 2 parts of aluminum stearate, mix them evenly in an oil phase tank, heat and melt until semi-transparent, add 6 parts of Span 80, and maintain at a temperature of 125 - 130 °C for 30 min, then cool to room temperature to obtain the oil phase;

[0059] S2. Preparation of water phase

[0060] Take 4 parts of sterilized Tween 80 and add 96 parts of the qualified semi-finished product, stir well until Tween 80 is completely dissolved to obtain the water phase;

[0061] S3. Emulsification

[0062] Take 2 parts of the oil phase and place it in a high-speed shearing machine. Start the motor and stir at low speed, while slowly adding 1 part of the water phase, then emulsify at 3600 r / min for 40 min, quantitatively dispense, and seal the bottle mouth to obtain the Riemerella anatipestifer complex vaccine.

[0063] After emulsification is completed, take 10 ml of the sample and add it to a centrifuge tube, centrifuge at 3000 r / min for 15 min, and there should be no water phase precipitation at the bottom of the tube.

[0064] Example 2

[0065] In vitro expression and identification of recombinant PVAX1-OmpA plasmid

[0066] Select well-grown PK-15 cells and inoculate them into a 6-well cell culture plate. When the cells reach 85-90% confluence in a 37 °C, 5% carbon dioxide incubator, perform transfection with the recombinant PVAX1-OmpA plasmid; the cell transfection operation is carried out according to the Lipofectamine TM 2000 kit instructions as follows:

[0067] S1. Take 4 µg of the recombinant PVAX1-OmpA plasmid and dilute it to 250 µl with serum-free cell culture medium, and gently mix to obtain a DNA suspension;

[0068] S2. Take 10 µl of Lipo2000, dilute it to 250 µl with serum-free cell culture medium, and gently mix, then let it stand at room temperature for 5 min;

[0069] S3. Mix the DNA suspension and the Lipo2000 suspension gently, and let it stand at room temperature for 20 min to obtain a mixed solution;

[0070] S4. Discard the culture medium in the 6-well plate where the PK-15 cells have grown well, wash it 2 times with serum-free cell culture medium, aspirate and discard the culture medium, add the mixed solution, gently mix, then supplement with 1 ml of serum-free cell culture medium, and place it in a 37 °C, 5% CO₂ incubator for culture;

[0071] S5. After 6 h of transfection, discard the cell fluid in the 6-well plate, and add 2 ml of DMEM medium containing 10% newborn bovine serum to each well;

[0072] S6. Set the PK-15 cells transfected with the pVAX1 empty vector as a negative control;

[0073] S7. After 48 h of transfection, wash the cell plate after virus inoculation 1 time with PBS solution at pH 7.2 gently to prevent cell detachment, fix the cells with pre-cooled methanol at 4 °C for 15-20 min, wash 3 times with PBS, add 100 µl of rabbit anti-Riemerella anatipestifer OmpA protein positive serum, incubate at 37 °C for 1 h, wash 3 times with PBS, add 100 µl of FITC-labeled goat anti-rabbit secondary antibody diluted 100-fold, incubate at 37 °C in the dark for 45 min, wash 3 times with PBS, and observe the results under a fluorescence microscope;

[0074] Results: Obvious fluorescence signals can be observed after the recombinant PVAX1-OmpA plasmid is transfected into PK-15 cells, while there are no obvious fluorescence signals in the control group, proving that the constructed recombinant PVAX1-OmpA plasmid can correctly express the OmpA protein of Riemerella anatipestifer.

[0075] Example 3

[0076] Detection of the recombinant PVAX1-OmpA plasmid:

[0077] 1. Determination of the concentration of the recombinant PVAX1 - OmpA plasmid: Use a ultra - micro nucleic acid analyzer to detect the plasmid concentration, which should be not less than 500 µg / ml.

[0078] 2. Enzyme digestion identification: Digest the purified recombinant PVAX1 - OmpA plasmid with Kpn I and Xho I double enzymes and then perform agarose gel electrophoresis analysis; two bands of approximately 3000 bp and 1164 bp in size should be seen in the result.

[0079] 3. Host protein detection: Prepare a standard curve with BSA of known concentration according to the instructions of the BCA protein detection kit; dilute the purified recombinant PVAX1 - OmpA plasmid with sterile water in gradients and quantitatively detect the bacterial proteins in the purified plasmid DNA under the same conditions. The content of bacterial proteins in the recombinant PVAX1 - OmpA plasmid is less than 10 µg / mg.

[0080] 4. Bacterial endotoxin detection: Perform endotoxin detection according to the Limulus reagent method, and the endotoxin content should be less than 1000 EU / mg.

[0081] Example 4

[0082] Inspection of the Riemerella anatipestifer complex vaccine:

[0083] (I). Physical properties

[0084] 1. The appearance is a milky white emulsion.

[0085] 2. The dosage form is water - in - oil type. Take a clean pipette and suck a small amount of the vaccine and drop it into cold water. Except for the first drop, it does not spread.

[0086] 3. Stability: Suck 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 min. No aqueous phase precipitates at the bottom of the tube.

[0087] 4. Viscosity: Conduct according to the current "Chinese Veterinary Pharmacopoeia" and meet the requirements.

[0088] 5. Inspection of filling volume: Conduct according to the current "Chinese Veterinary Pharmacopoeia" and meet the requirements.

[0089] (II). Sterility test

[0090] Conduct according to the current "Chinese Veterinary Pharmacopoeia", and there is no bacterial growth.

[0091] (III). Determination of formaldehyde residue

[0092] Conduct according to the current "Chinese Veterinary Pharmacopoeia" and meet the requirements.

[0093] Example 5

[0094] Study on the Immunization Program of Riemerella anatipestifer Combined Vaccine for Laying Hens

[0095] (I) Experimental grouping: 150 laying hens at 380 days old were selected and evenly divided into 5 groups, with 30 hens in each group, namely the combined vaccine group, the capsular polysaccharide group, the DNA vaccine group, the whole cell inactivated vaccine group, and the non-immunized control group; the immunogen of the combined vaccine group was the Riemerella anatipestifer combined vaccine prepared in Example 1, the immunogen of the capsular polysaccharide group was the capsular polysaccharide solution prepared in Example 1 emulsified with white oil adjuvant at a ratio of 1:2 after inactivated with formaldehyde, and the final concentration of capsular polysaccharide in the aqueous phase was 0.3 mg / ml; the immunogen of the DNA vaccine group was the recombinant PVAX1-OmpA plasmid prepared in Example 1, diluted to 20 μg / ml with 10 mmol / L TE buffer; the immunogen of the whole cell inactivated vaccine group was the Riemerella anatipestifer whole cell inactivated vaccine, and its preparation method was to ferment and prepare the Riemerella anatipestifer fermentation broth according to the method of steps (1)-(3) of Example 1, centrifuge to discard the supernatant, resuspend the cell precipitate with original strength physiological saline, inactivate with formaldehyde and then emulsify with white oil adjuvant at a ratio of 1:2; the non-immunized control group was injected with the same volume of physiological saline.

[0096] (II) Method: Each group of laying hens was immunized with the corresponding vaccine by subcutaneous injection at 0.5 ml / hen, with an immunization interval of 14 days, and a total of three immunizations. Eggs were collected before immunization, 14 days after the second immunization, and 7, 14, 21, and 28 days after the third immunization to determine the egg yolk agar gel diffusion antibody titer. After that, high-titer eggs were collected every 30 days to determine the egg yolk agar gel diffusion antibody titer, and the egg yolk antibody production period and persistence period of laying hens were determined.

[0097] (III) Results: After immunizing laying hens with the prepared Riemerella anatipestifer combined vaccine, the antibody titer could reach 1:64 on the 7th day after the third immunization (see Table 1), and the highest could reach 1:128. Moreover, when detected 2 months after the third immunization, the egg yolk antibody titer of eggs reached 1:32, still higher than the egg collection standard (not less than 1:32) (see Table 2); while the egg yolk antibody titers of other groups did not reach 1:32; at the same time, the Riemerella anatipestifer combined vaccine, the capsular polysaccharide vaccine, and the DNA vaccine had no obvious effect on the egg production rate of immunized laying hens, while the whole cell inactivated vaccine group caused a serious decline in the egg production rate of laying hens after the third immunization (see Table 3); in summary, whether it is the egg yolk antibody production time, antibody production level, antibody persistence period or the impact on the egg production rate of laying hens, the combined vaccine is far superior to the capsular polysaccharide vaccine, the DNA vaccine, and the whole cell inactivated vaccine, showing good application prospects.

[0098] Table 1 Egg yolk antibody production period of laying hens after immunization in each group

[0099]

[0100] Table 2 Duration of yolk antibody in laying hens after immunization in each group

[0101]

[0102] Table 3 Laying performance changes of laying hens after immunization in each group

[0103]

[0104] Example 6 Preparation and efficacy experiment of yolk antibody against Riemerella anatipestifer

[0105] (I). Preparation of yolk antibody against Riemerella anatipestifer

[0106] Laying hens were immunized with a combined vaccine of Riemerella anatipestifer according to the method of Example 5 to prepare hyperimmune eggs. Qualified hyperimmune eggs (antibody titer not less than 1:32) were collected. After disinfecting the eggshells, yolk separation, octanoic acid extraction, inactivation, filtration and sterilization, and sub-packaging steps were carried out (the octanoic acid extraction method is a conventional extraction method and will not be elaborated here). Yolk antibody against Riemerella anatipestifer was prepared, and the antibody titer of the final product was not less than 1:4;

[0107] (II). Safety experiment of yolk antibody against Riemerella anatipestifer

[0108] Twenty 14-day-old healthy and susceptible ducklings were selected and subcutaneously injected with the yolk antibody against Riemerella anatipestifer prepared in step (I) at a dose of 2.0 ml per duckling. After immunization, they were observed for 14 days. All the ducklings survived and showed no adverse reactions, indicating that the yolk antibody against Riemerella anatipestifer prepared in step (I) has good safety.

[0109] Example 7 Efficacy experiment of yolk antibody against Riemerella anatipestifer

[0110] (I). Method: Ninety 14-day-old healthy and susceptible ducklings were evenly divided into three groups, with 30 ducklings in each group. The first group was the yolk antibody treatment group, which was intramuscularly injected with Riemerella anatipestifer at a dose of 1.0×10 8.0 CFU per duckling, and 24 hours later, subcutaneously injected with the yolk antibody against Riemerella anatipestifer prepared in Example 6 at a dose of 0.5 ml per duckling; the second group was the yolk antibody prevention group, which was subcutaneously injected with the yolk antibody against Riemerella anatipestifer at a dose of 0.5 ml per duckling, and 24 hours later, intramuscularly injected with a virulent strain of Riemerella anatipestifer at a dose of 1.0×10 8.0 CFU per duckling; the third group was the normal saline control group, which was intramuscularly injected with a virulent strain of Riemerella anatipestifer at a dose of 1.0×10 8.0 CFU per duckling, and 24 hours later, subcutaneously injected with sterile normal saline at a dose of 0.5 ml per duckling; After challenge, the ducklings were continuously observed for 7 days, and the death of ducklings was used as the criterion for judging the onset of Riemerella anatipestifer;

[0111] (2) Results: All 30 ducklings in the yolk antibody prevention group survived healthily, achieving 100% protection. Among the ducklings in the yolk antibody treatment group, 26 survived, with an antibody protection rate of 86.7%. 90% of the ducklings in the normal saline control group became ill, and 27 died.

[0112] Example 8

[0113] The difference between this example and Example 1 is that in the first step of step (3), the final concentration of capsular polysaccharide in the capsular polysaccharide solution is 0.1 mg / ml, and the final concentration of the recombinant PVAX1 - OmpA plasmid is 10 µg / ml, with the remaining operations being the same.

[0114] Example 9

[0115] The difference between this example and Example 1 is that in the first step of step (3), the final concentration of capsular polysaccharide in the capsular polysaccharide solution is 0.6 mg / ml, and the final concentration of the recombinant PVAX1 - OmpA plasmid is 100 µg / ml, with the remaining operations being the same.

[0116] This application provides a preparation method of a Riemerella anatipestifer composite vaccine and yolk antibody. First, the capsular polysaccharide of Riemerella anatipestifer is extracted by the CTAB method, and then a recombinant PVAX1 - OmpA plasmid is prepared, which is formed by enzymatic digestion and ligation of the OmpA gene of Riemerella anatipestifer and the eukaryotic expression vector PVAX1; the extracted capsular polysaccharide of Riemerella anatipestifer and the recombinant PVAX1 - OmpA plasmid are mixed to prepare an aqueous phase, and the aqueous phase and an oil phase are emulsified to obtain the Riemerella anatipestifer composite vaccine; after immunizing laying hens with the composite vaccine, the egg collection requirement can be met 7 days after the third immunization, with a maximum of 1:128. In terms of the yolk antibody production time, antibody production level, and antibody persistence period, it is significantly superior to the whole - cell inactivated vaccine; and when detected 2 months after the third immunization, the yolk antibody titer still meets the requirements. All ducklings in the yolk antibody prevention group of Riemerella anatipestifer survived healthily, achieving 100% protection. The antibody protection rate of ducklings in the yolk antibody treatment group was 86.7%, while 90% of the ducklings in the normal saline control group became ill; the results show that the yolk antibody prepared by the present invention has a good clinical protection effect on ducklings infected with Riemerella anatipestifer and can be used for the prevention and treatment of Riemerella anatipestifer; in addition, the technical solution provided by this application can also reduce the preparation cost of the yolk antibody of Riemerella anatipestifer, reduce the immune stress of laying hens, and has great popularization and application value.

[0117] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of this application, "a plurality" means two or more, unless otherwise specifically defined.

[0118] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A preparation method of a Riemerella anatipestifer composite vaccine, characterized in that, It includes the following steps: (1) Mix the capsular polysaccharide solution and the recombinant PVAX1-OmpA plasmid in proportion, and inactivate to obtain a semi-finished product; The preparation method of the capsular polysaccharide solution is as follows: ferment and culture Riemerella anatipestifer to obtain a fermentation broth, centrifuge the fermentation broth to obtain the supernatant, add cetyltrimethylammonium bromide solution and then centrifuge to discard the supernatant, resuspend the precipitate with sodium chloride solution, add ethanol and then centrifuge to discard the supernatant, dissolve the precipitate to obtain the capsular polysaccharide solution; the final concentration of the Riemerella anatipestifer fermentation broth after adding the cetyltrimethylammonium bromide solution is 5 mM; the concentration of the sodium chloride solution is 0.4 M, and the concentration of the ethanol is 95%; The preparation method of the recombinant PVAX1-OmpA plasmid is as follows: (1) Construction of the vector of the recombinant PVAX1-OmpA plasmid: the outer membrane protein OmpA gene of Riemerella anatipestifer is synthesized by total gene synthesis, and the outer membrane protein OmpA gene is ligated with the eukaryotic expression vector PVAX1 by restriction enzyme digestion to construct the recombinant PVAX1-OmpA plasmid, and it is transferred into Escherichia coli DH5α; (2) Large-scale preparation of the recombinant PVAX1-OmpA plasmid: after fermenting and culturing the Escherichia coli DH5α, centrifuge to collect the bacterial cells, successively add hydrochloric acid buffer solution, sodium hydroxide solution and acetic acid solution, and perform post-treatment to obtain a purified recombinant PVAX1-OmpA plasmid solution; remove the bacterial endotoxin of the purified recombinant PVAX1-OmpA plasmid solution to obtain a large-scale prepared recombinant PVAX1-OmpA plasmid; In step (2), the ratio of the bacterial cells to the hydrochloric acid buffer solution is 1 g : 5 ml, and the volume ratio of the hydrochloric acid buffer solution, the sodium hydroxide solution and the acetic acid solution is 1 : 2 : 1.5; The final concentration of the capsular polysaccharide in the capsular polysaccharide solution is 0.1 - 0.6 mg / ml, and the final concentration of the recombinant PVAX1-OmpA plasmid is 10 - 100 μg / ml; (2) Mix the white oil for injection and aluminum stearate, heat and then add Span 80, raise the temperature and mix to obtain an oil phase; mix Tween 80 and the semi-finished product to obtain an aqueous phase; mix the oil phase and the aqueous phase for emulsification to obtain a Riemerella anatipestifer combined vaccine.

2. The preparation method of a Riemerella anatipestifer complex vaccine according to claim 1, characterized in that, In step (1), the precipitate is dissolved with physiological saline; the inactivation is carried out with formaldehyde solution.

3. The preparation method of a Riemerella anatipestifer composite vaccine according to claim 1, characterized in that, The formula of the hydrochloric acid buffer solution is 25 mM Tris-HCl, 10 mM EDTA, 50 mM glucose; the formula of the sodium hydroxide solution is 250 mM NaOH, 1% SDS; the formula of the acetic acid solution is 3 M potassium acetate, 5 M acetic acid.

4. The preparation method of a Riemerella anatipestifer complex vaccine according to claim 1, characterized in that, By weight, the white oil for injection is 94 parts, aluminum stearate is 2 parts, Span 80 is 6 parts, Tween 80 is 4 parts, the semi-finished product is 96 parts, the oil phase is 2 parts, and the aqueous phase is 1 part.

5. A preparation method of Riemerella anatipestifer yolk antibody, characterized in that, Specifically, it includes the following steps: Use the combined vaccine prepared by the preparation method of the Riemerella anatipestifer combined vaccine according to any one of claims 1 - 4 to immunize laying hens, collect eggs after immunization to obtain high-immune eggs, and extract the high-immune eggs to obtain Riemerella anatipestifer yolk antibody.

6. The preparation method of a Riemerella anatipestifer yolk antibody according to claim 5, characterized in that, The vaccine is immunized three times, with an interval of 14 days each time, and the injection volume of the vaccine is 0.5 ml per animal.

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