Riemerella anatipestifer composite vaccine and preparation method of egg yolk antibody

Abstract

The invention discloses a riemerella anatipestifer composite vaccine and a preparation method of an egg yolk antibody, and belongs to the technical field of biology. Riemerella anatipestifer capsular polysaccharide and recombinant PVAX1-OmpA plasmids are mixed to prepare the riemerella anatipestifer composite vaccine; after laying hens are immunized by the composite vaccine, the egg collection requirement can be met 7 days after three immunization, and the egg yolk antibody production time, the antibody production height and the antibody duration of the composite vaccine are obviously superior to those of a whole-cell inactivated vaccine; the titer of the egg yolk antibody still meets the requirement after detection is carried out for 2 months after three immunization, and the egg yolk antibody has a good clinical protection effect on ducklings infected by riemerella anatipestifer and can be used for preventing and treating riemerella anatipestifer; the method also can reduce the preparation cost of the riemerella anatipestifer egg yolk antibody, can reduce the immune stress of laying hens, and has great popularization and application values.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and in particular relates to a method for preparing a Riemerella anatipestifer composite vaccine and egg yolk antibodies. Background Art

[0002] Riemerella anatipestifer is a Gram-negative rod-shaped bacterium with no flagella, no spores, and can form capsules. It is a facultative anaerobe and can grow on tryptone soy agar, blood agar, and chocolate agar. Riemerella anatipestifer infects multiple duck breeds, including Cherry Valley ducks, Mallard ducks, and Muscovy ducks. Ducks aged 1-5 weeks are most susceptible. The clinical symptoms of the diseased ducks first manifest as depression, reduced feed intake, tears, sneezing, and green loose feces, followed by ataxia and head and neck tremors. The number of ducks with neurological symptoms in the later stage increases significantly. After autopsy, the diseased ducks mainly show fibrinous exudation on the serosal surface of the organs, which is most obvious in the heart and liver. The morbidity and mortality rates range from 5-75%. The surviving ducks that survive the infection grow slowly and weigh significantly less than normal ducks. Some ducks have sequelae of encephalitis and become "stiff ducks" or "residual ducks." Riemerella anatipestifer has a wide range of prevalence, high infection rate and mortality rate, causing huge economic losses to the duck industry. It is one of the most common diseases that endanger duck farming.

[0003] Due to the serious drug resistance of Riemerella anatipestifer, the number of clinical cases of failed antibiotic treatment has increased year by year. At the same time, antibiotic treatment of Riemerella anatipestifer will cause a series of problems such as food safety and environmental pollution. Therefore, how to develop more effective antibiotic reduction and replacement products for the prevention and control of Riemerella anatipestifer is a hot topic in current research. Yolk antibodies are a type of specific IgY antibody extracted from high-immunity eggs produced after immunization of laying hens. They are often used in the prevention and control of clinical viral and bacterial diseases. They are cheap, green and environmentally friendly, highly specific, and effective. Riemerella anatipestifer is a Gram-negative bacterium. The bacteria contain a large amount of bacterial endotoxins. Directly making a whole-bacterial inactivated vaccine to immunize laying hens with multiple injections can easily damage the oviducts of laying hens and reduce egg production, resulting in a lag in the development of Riemerella anatipestifer yolk antibodies. Summary of the invention

[0004] The purpose of the present application is to provide a method for preparing a composite vaccine of Riemerella anatipestifer and egg yolk antibodies, so as to solve the technical problems existing in the prior art of excessive side effects, poor effects and short duration of whole-bacteria inactivated vaccines.

[0005] To achieve the above object, the technical solution adopted in the present application is: to provide a method for preparing a Riemerella anatipestifer composite vaccine, comprising the following steps: (i) mixing the capsular polysaccharide solution and the recombinant PVAX1-OmpA plasmid in proportion, and inactivating the mixture to obtain a semi-finished product; (ii) Mix white oil for injection and aluminum stearate, add Tween 80 after heating, and mix by heating 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 composite vaccine.

[0006] In one embodiment, The preparation method of the capsular polysaccharide solution is as follows: fermenting and culturing Riemerella anatipestifer to obtain a fermentation broth, centrifuging the fermentation broth to obtain a supernatant, adding a hexadecyltrimethylammonium bromide solution and then centrifuging to discard the supernatant, resuspending the precipitate with a sodium chloride solution, adding ethanol and then centrifuging to discard the supernatant, dissolving the precipitate to obtain a capsular polysaccharide solution.

[0007] In one embodiment, The final concentration of the fermentation broth of Riemerella anatipestifer after adding the hexadecyltrimethylammonium 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; and the inactivation is carried out with formaldehyde solution.

[0008] In one embodiment, The preparation method of recombinant PVAX1-OmpA plasmid is as follows: (I) Construction of recombinant PVAX1-OmpA plasmid vector: synthesize the outer membrane protein OmpA gene of Riemerella anatipestifer, connect the outer membrane protein OmpA gene with the eukaryotic expression vector PVAX1 through enzyme digestion to construct a recombinant PVAX1-OmpA plasmid, and transfer it into Escherichia coli DH5α; (ii) Large-scale preparation of recombinant PVAX1-OmpA plasmid: After the Escherichia coli DH5α is fermented and cultured, the bacteria are collected by centrifugation, and hydrochloric acid buffer, sodium hydroxide solution and acetic acid solution are added in sequence, and post-treatment is performed to obtain a purified recombinant PVAX1-OmpA plasmid solution; bacterial endotoxins are removed from the purified recombinant PVAX1-OmpA plasmid solution to obtain a large-scale prepared recombinant PVAX1-OmpA plasmid.

[0009] In one embodiment, In step (ii), 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.

[0010] In one embodiment, The formula of the hydrochloric acid buffer solution is 25 mM Tris-HCl, 10 mM EDTA, and 50 mM glucose; the formula of the sodium hydroxide solution is 250 mM NaOH and 1% SDS; and the formula of the acetic acid solution is 3 M potassium acetate and 5 M acetic acid.

[0011] In one embodiment, 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. Preferably, the final concentration of the capsular polysaccharide is 0.3 mg / ml, and the final concentration of the recombinant PVAX1-OmpA plasmid is 50 µg / ml.

[0012] In one embodiment, In parts by weight, the white oil for injection is 94 parts, aluminum stearate is 2 parts, Siben 80 is 6 parts, Tween 80 is 4 parts, the semi-finished product is 96 parts, the oil phase is 2 parts, and the water phase is 1 part.

[0013] The present application also provides a method for preparing Riemerella anatipestifer yolk antibodies, which specifically comprises the following steps: immunizing laying hens with the composite vaccine prepared by the method for preparing the Riemerella anatipestifer composite vaccine of any of the above embodiments, collecting eggs after immunization to obtain high-immune eggs, separating the yolks of the high-immune eggs, extracting with caprylic acid, inactivating, filtering and sterilizing, and packaging to obtain Riemerella anatipestifer yolk antibodies.

[0014] In one embodiment, The vaccine was administered three times, with an interval of 14 days between each immunization. The injection volume of the vaccine was 0.5 ml per animal.

[0015] The present application provides a method for preparing a composite vaccine and yolk antibodies of Riemerella anatipestifer. After immunizing laying hens with the composite vaccine, the egg collection requirements can be met 7 days after three immunizations. The composite vaccine is significantly superior to the whole-bacteria inactivated vaccine in terms of the time of yolk antibody production, the height of antibody production and the duration of antibody production. Moreover, the yolk antibody titer still meets the requirements when tested 2 months after the three immunizations. The yolk antibody has a good clinical protective effect on ducklings infected with Riemerella anatipestifer, and can be used for the prevention and treatment of Riemerella anatipestifer. The present method can also reduce the preparation cost of Riemerella anatipestifer yolk antibodies, reduce the immune stress of laying hens, and has great promotion and application value. DETAILED DESCRIPTION

[0016] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear, the present application is further described in detail. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0017] Riemerella anatipestifer was obtained commercially.

[0018] Example 1 1. Preparation of Capsular Polysaccharide Solution 1. Preparation of seeds for production The seeds of Riemerella anatipestifer were streaked onto chocolate nutrient agar plates and cultured in a candle jar at 37°C for 16-24 hours. Ten typical colonies were selected and inoculated into 100 ml of LB culture medium and cultured at 37°C for 8-10 hours as seed solution. 2. Preparation of culture medium The fermentation medium was tryptone soy broth medium, containing 17 g tryptone, 3 g soy peptone, 5 g sodium chloride, 2.5 g glucose, and 2.5 g dipotassium hydrogen phosphate per 1000 ml medium; 3. Bacterial culture Fill the culture tank with an appropriate amount of culture medium (accounting for 60-80% of the volume of the culture tank) and defoaming agent according to the volume of the culture tank, inoculate the seed solution according to 1% of the volume of the culture medium after sterilization, adjust the rotation speed and pH value to keep the dissolved oxygen above 60% and the pH value at about 7.2, and culture at 37°C for 6-8 hours with aeration to obtain the fermentation liquid; 4. Preparation of Riemerella anatipestifer Capsular Polysaccharide Solution S1. Centrifuge the fermentation liquid at 8000 r / min for 10 min, discard the bacterial precipitate, add CTAB solution with a final concentration of 5 mmol / L to the supernatant, mix well and refrigerate 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 times the volume of 95% ethanol, mix well and let stand at 4°C overnight, centrifuge at 8000 r / min for 10 min, discard the supernatant, dissolve the precipitate with sterile physiological saline to obtain a Riemerella anatipestifer capsular polysaccharide solution; S2. The capsular polysaccharide content of Riemerella anatipestifer was determined by the anthrone method. The capsular polysaccharide content should not be less than 1 mg / ml. (II) Preparation of recombinant PVAX1-OmpA plasmid 1. Construction of recombinant PVAX1-OmpA plasmid vector S1. According to the OmpA gene sequence of Riemerella anatipestifer JL-RA3 strain on NCBI (GenBank Number: HQ707078), Kpn I and Xho I double restriction sites were added to both ends of the OmpA gene, and then the whole gene was synthesized to obtain the synthetic outer membrane protein OmpA gene; S2, the outer membrane protein OmpA gene was linked into the corresponding restriction site of the eukaryotic expression vector PVAX1, the plasmid was extracted and double-digested with Kpn I and Xho I for identification, and sequenced after it was correct (the sequencing results showed that the OmpA gene of Riemerella anatipestifer was successfully linked into the corresponding restriction site of the PVAX1 vector), and the recombinant PVAX1-OmpA plasmid was successfully constructed; CaCl 2The recombinant PVAX1-OmpA plasmid was transformed into Escherichia coli DH5α by the method; 2. Large-scale preparation of recombinant PVAX1-OmpA plasmid S1. High-density fermentation and bacterial lysis of recombinant bacteria After high-density fermentation of Escherichia coli DH5α containing recombinant PVAX1-OmpA plasmid DNA in a 50 L fermenter, the cells were collected by centrifugation at 5000 r / min for 10 min, 5 ml of hydrochloric acid buffer was added per 1 g of wet bacteria, and then hydrochloric acid buffer, sodium hydroxide solution and acetic acid solution were added at a volume ratio of 1:2:1.5, respectively. After incubation at room temperature for 15 min, the cells were centrifuged at 10000 r / min for 10 min at room temperature, the supernatant was collected, 0.7 times the volume of isopropanol was added, and the cells were precipitated at -20 °C for 30 min. The cells were centrifuged at 10000 r / min for 10 min at room temperature, the supernatant was discarded, and the precipitate was dissolved in 10 mmol / L TE buffer to obtain a purified plasmid DNA solution; The formula of hydrochloric acid buffer is 25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose; The formula of sodium hydroxide solution was 250 mM NaOH, 1% (w / v) SDS; The formula of acetic acid solution is 3 M potassium acetate, 5 M acetic acid; S2. Removal of bacterial endotoxins Add 10% TritonX-114 to the purified plasmid solution to make the final concentration 1%, mix well, place on ice for 10 min, continue incubation at 42°C for 10 min, centrifuge at 10000 r / min for 10 min at room temperature, carefully pipette the supernatant into a pyrogen-free container, and repeat the operation once if necessary; dilute to 500 µg / ml with 10 mmol / L TE buffer, and quantitatively dispense to obtain the recombinant PVAX1-OmpA plasmid; (III) Preparation of Riemerella anatipestifer composite vaccine 1. Preparation of semi-finished products The Riemerella anatipestifer capsular polysaccharide solution prepared in step (i) and the recombinant PVAX1-OmpA plasmid prepared in step (ii) were diluted and mixed to make the final concentration of the capsular polysaccharide 0.3 mg / ml and the final concentration of the DNA in the recombinant PVAX1-OmpA plasmid 50 µg / ml, and formaldehyde was added at a final concentration of 0.2%, and inactivated at 37°C for 24 h to obtain a semi-finished product; 2. Vaccine preparation S1. Oil phase preparation Take 94 parts of high-quality injection white oil and 2 parts of aluminum stearate, mix them evenly in an oil phase tank, heat and melt until they are translucent, add 6 parts of Siben 80, maintain the temperature at 125-130°C for 30 minutes, and cool to room temperature to obtain the oil phase; S2. Preparation of aqueous phase Take 4 parts of sterilized Tween 80, add 96 parts of the semi-finished product that has passed the inspection, and stir thoroughly until the Tween 80 is completely dissolved to prepare the water phase; S3, emulsification Take 2 portions of the oil phase and place them in a high-speed shearing machine, start the motor to stir at a low speed, and slowly add 1 portion of the water phase. Emulsify at 3600 r / min for 40 min, package quantitatively, seal the bottle mouth, and obtain the Riemerella anatipestifer composite vaccine.

[0019] After emulsification, take 10 ml of sample and add it to a centrifuge tube. Centrifuge at 3000 r / min for 15 min. There should be no water phase precipitated at the bottom of the tube.

[0020] Example 2 In vitro expression and identification of recombinant PVAX1-OmpA plasmid Well-growing PK-15 cells were selected and inoculated into 6-well cell culture plates. The cells were cultured in a 37°C, 5% carbon dioxide incubator until the cells were 85-90% confluent, and then the recombinant PVAX1-OmpA plasmid was transfected. The cell transfection operation was performed according to Lipofectamine TM 2000 kit instructions, the steps are as follows: S1. Take 4 µg of recombinant PVAX1-OmpA plasmid and dilute it to 250 µl with serum-free cell culture medium. Mix gently to prepare DNA suspension. S2. Take 10 µl of Lipo2000, dilute to 250 µl with serum-free cell culture medium, mix gently, and let stand at room temperature for 5 min. S3, mix the DNA suspension and Lipo2000 suspension, gently mix, and let stand at room temperature for 20 min to obtain a mixed solution; S4. Discard the culture medium in the 6-well plate where PK-15 cells have grown, wash twice with serum-free cell culture medium, discard the culture medium, add the mixed solution, mix gently, add 1 ml serum-free cell culture medium, and place at 37 °C and 5% CO. 2 Culture in an incubator; S5. 6 h after transfection, discard the cell solution in the 6-well plate and add 2 ml of DMEM medium containing 10% newborn calf serum to each well; S6, PK-15 cells transfected with pVAX1 empty vector were used as negative control; S7. After 48 hours of transfection, wash the infected cell plate once with PBS solution at pH 7.2, gently to prevent the cells from falling off, 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 hour, wash 3 times with PBS, add 100 µl of 100-fold diluted FITC-labeled goat anti-rabbit secondary antibody, incubate at 37°C in the dark for 45 min, wash 3 times with PBS, and observe the results under a fluorescence microscope; Results Obvious fluorescence signals were observed after recombinant PVAX1-OmpA plasmid was transfected into PK-15 cells, while no obvious fluorescence signals were observed in the control group, which proved that the constructed recombinant PVAX1-OmpA plasmid could correctly express the OmpA protein of Riemerella anatipestifer.

[0021] Example 3 Detection of recombinant PVAX1-OmpA plasmid: 1. Determination of the concentration of recombinant PVAX1-OmpA plasmid: The concentration of the plasmid was detected by ultra-micro nucleic acid analyzer and was not less than 500 µg / ml.

[0022] 2. Enzyme digestion identification: The purified recombinant PVAX1-OmpA plasmid was double digested with Kpn I and Xho I and analyzed by agarose gel electrophoresis; the result should show two bands of approximately 3000 bp and 1164 bp in size; 3. Host protein detection: According to the instructions of the BCA protein detection kit, a standard curve was prepared with BSA of known concentration; the purified recombinant PVAX1-OmpA plasmid was gradiently diluted with sterile water, and the bacterial protein in the purified plasmid DNA was quantitatively detected under the same conditions. The bacterial protein content of the recombinant PVAX1-OmpA plasmid was less than 10 µg / mg; 4. Bacterial endotoxin test: Endotoxin test shall be conducted according to the horseshoe crab reagent method. The endotoxin content shall be less than 1000 EU / mg.

[0023] Example 4 Inspection of Riemerella anatipestifer compound vaccine: 1. Physical properties 1. The appearance is milky white emulsion; 2. The dosage form is oil-in-water type. Take a clean pipette and draw a small amount of vaccine and drop it into cold water. Except for the first drop, it will not spread; 3. Stability: Pipette 10 ml of the vaccine into a centrifuge tube and centrifuge at 3000 r / min for 15 min. No water phase will precipitate at the bottom of the tube. 4. Viscosity: According to the current "Chinese Veterinary Pharmacopoeia", in compliance with regulations; 5. Filling quantity inspection: It shall be carried out in accordance with the current "Chinese Veterinary Pharmacopoeia" and meet the regulations; 2. Sterility test According to the current "Chinese Veterinary Pharmacopoeia", sterile growth; (III) Determination of residual formaldehyde It is carried out in accordance with the current "Chinese Veterinary Pharmacopoeia" and complies with regulations.

[0024] Example 5 Study on the immunization program of laying hens with Riemerella anatipestifer compound vaccine: (I) Experimental grouping: 150 laying hens aged 380 days were selected and divided into 5 groups, 30 in each group, including a composite vaccine group, a capsular polysaccharide group, a DNA vaccine group, a whole-bacterial inactivated vaccine group, and a non-immunized control group; the immunogen of the composite vaccine group was the Riemerella anatipestifer composite vaccine prepared in Example 1, the immunogen of the capsular polysaccharide group was the capsular polysaccharide solution prepared in Example 1, which was inactivated by formaldehyde and emulsified with white oil adjuvant at a ratio of 1:2, wherein 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, which was diluted to 20 μl with 10 mmol / L TE buffer. μg / ml; the whole cell inactivated vaccine group's immunogen was a whole cell inactivated vaccine of Riemerella anatipestifer, which was prepared by fermenting a Riemerella anatipestifer fermentation broth according to the method 1-3 of step (i) of Example 1, centrifuging and discarding the supernatant, resuspending the bacterial precipitate with the original volume of physiological saline, inactivating it with formaldehyde, and emulsifying it with a white oil adjuvant at a ratio of 1:2; the non-immunized control group was injected with an equal volume of physiological saline.

[0025] 2. Methods: Each group of laying hens were immunized with corresponding vaccines by subcutaneous injection, 0.5 ml / hen, with an interval of 14 days between immunizations, for 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 titer of yolk agar-agar-expanded antibodies. After that, high-immunity eggs were collected every 30 days to determine the titer of yolk agar-agar-expanded antibodies to determine the yolk antibody production period and duration period of laying hens.

[0026] (III) Results: After immunizing laying hens with the prepared Riemerella anatipestifer compound vaccine, the antibody titer could reach 1:64 (see Table 1) 7 days after the three immunizations, and the highest could reach 1:128. In addition, the yolk antibody titer of the eggs was tested 2 months after the three immunizations, reaching 1:32, which was still higher than the egg collection standard (not less than 1:32) (see Table 2); while the yolk antibody titers of the other groups did not reach 1:32; at the same time, the Riemerella anatipestifer compound vaccine, capsular polysaccharide vaccine, and DNA vaccine had no significant effect on the egg production rate of the immunized laying hens, while the whole-bacteria inactivated vaccine group caused a serious decrease in the egg production rate of laying hens after the three immunizations (see Table 3); in summary, whether it is the time of yolk antibody production, the height of antibody production, the duration of antibody, or the effect on the egg production rate of laying hens, the compound vaccine is far superior to the capsular polysaccharide vaccine, DNA vaccine, and whole-bacteria inactivated vaccine, showing a good application prospect.

[0027] Table 1 Yolk antibody production period of laying hens in each group after immunization

[0028] Table 2 The duration of yolk antibody in laying hens after immunization in each group

[0029] Table 3 Changes in egg production of laying hens after immunization in each group

[0030] Example 6 Preparation and efficacy experiment of Riemerella anatipestifer egg yolk antibody 1. Preparation of Riemerella anatipestifer yolk antibodies According to the method of Example 5, laying hens were immunized with the Riemerella anatipestifer composite vaccine to prepare high-immune eggs, and qualified high-immune eggs (antibody titer was not less than 1:32) were collected. After the eggshell was disinfected, yolk separation, caprylic acid extraction, inactivation, filtration sterilization, and subpackaging steps were performed (caprylic acid extraction method is a conventional extraction method and will not be repeated here), and Riemerella anatipestifer yolk antibodies were obtained. The antibody titer of the final product was not less than 1:4; 2. Safety test of Riemerella anatipestifer egg yolk antibody Twenty healthy susceptible ducklings of 14 days old were selected and injected subcutaneously into the neck with 2.0 ml / duck of the Riemerella anatipestifer yolk antibody prepared in step (I). After 14 days of observation after immunization, all the ducklings were alive and well without any adverse reactions, indicating that the Riemerella anatipestifer yolk antibody prepared in step (I) has good safety.

[0031] Example 7 Efficacy experiment of Riemerella anatipestifer egg yolk antibody 1. Methods: 90 healthy susceptible ducklings aged 14 days were divided into three groups, 30 in each group. The first group was the egg yolk antibody treatment group, and the leg muscles were injected with Riemerella anatipestifer at 1.0×10 8.0 CFU / mouse, and 24 hours later, the yolk antibody of Riemerella anatipestifer prepared in Example 6 was injected subcutaneously in the neck, 0.5 ml / mouse; the second group was the yolk antibody prevention group, which was injected subcutaneously in the neck with Riemerella anatipestifer yolk antibody, 0.5 ml / mouse, and 24 hours later, the leg muscle was injected with Riemerella anatipestifer virulent, 1.0×10 8.0 CFU / each; the third group was the normal saline control group, and the leg muscles were injected with Riemerella anatipestifer virulent, 1.0×10 8.0 CFU / duck, 0.5 ml / duck was injected subcutaneously in the neck after 24 hours; ducklings were observed for 7 consecutive days after the challenge, and the death of ducklings was used as the criterion for the occurrence of Riemerella anatipestifer. 2. Results: All 30 ducklings in the egg yolk antibody prevention group were alive and well, with 100% protection. 26 ducklings in the egg yolk antibody treatment group were alive and well, with an antibody protection rate of 86.7%. 90% of the ducklings in the saline control group became ill, and 27 died.

[0032] Example 8 The difference between this example and example 1 is that in step (III) step 1, the final concentration of the capsular polysaccharide in the capsular polysaccharide solution is 0.1 mg / ml, the final concentration of the recombinant PVAX1-OmpA plasmid is 10 µg / ml, and the rest of the operations are the same.

[0033] Example 9 The difference between this example and example 1 is that in step (III) step 1, the final concentration of the capsular polysaccharide in the capsular polysaccharide solution is 0.6 mg / ml, the final concentration of the recombinant PVAX1-OmpA plasmid is 100 µg / ml, and the rest of the operations are the same.

[0034] The present application provides a method for preparing a composite vaccine of Riemerella anatipestifer and egg yolk antibodies, comprising the following steps: firstly, extracting Riemerella anatipestifer capsular polysaccharide by CTAB method, and then preparing a recombinant PVAX1-OmpA plasmid, which is formed by connecting the Riemerella anatipestifer OmpA gene and the eukaryotic expression vector PVAX1 by enzyme cutting; mixing the extracted Riemerella anatipestifer capsular polysaccharide with the recombinant PVAX1-OmpA plasmid to prepare an aqueous phase, and emulsifying the aqueous phase and the oil phase to prepare a composite vaccine of Riemerella anatipestifer; after immunizing laying hens with the composite vaccine, the egg collection requirement can be met 7 days after three immunizations, with the highest ratio reaching 1:128, and the composite vaccine is significantly superior to the whole-bacterial inactivated vaccine in terms of egg yolk antibody production time, antibody production height and antibody duration; and the egg yolk antibody titer still meets the requirements when tested 2 months after the three immunizations, and the ducklings in the Riemerella anatipestifer egg yolk antibody prevention group are all alive and well, obtaining 100% protection, and the antibody protection rate of the ducklings in the egg yolk antibody treatment group is 86.7 %, while 90% of the ducklings in the normal saline control group became ill; the results show that the egg yolk antibody prepared by the present invention has a good clinical protective 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 the present application can also reduce the preparation cost of Riemerella anatipestifer egg yolk antibodies, reduce the immune stress of laying hens, and has great promotion and application value.

[0035] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0036] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a Riemerella anatipestifer composite vaccine, characterized in that: The following steps are involved: (i) mixing the capsular polysaccharide solution and the recombinant PVAX1-OmpA plasmid in proportion, and inactivating the mixture to obtain a semi-finished product; (ii) Mix white oil for injection and aluminum stearate, add Tween 80 after heating, and mix by heating 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 composite vaccine.

2. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 1, characterized in that: The preparation method of the capsular polysaccharide solution comprises the following steps: fermenting and culturing Riemerella anatipestifer to obtain a fermentation broth, centrifuging the fermentation broth to obtain a supernatant, adding a hexadecyltrimethylammonium bromide solution and then centrifuging to discard the supernatant, resuspending the precipitate with a sodium chloride solution, adding ethanol and then centrifuging to discard the supernatant, dissolving the precipitate and obtaining a capsular polysaccharide solution.

3. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 2, characterized in that: The final concentration of the fermentation broth of Riemerella anatipestifer after adding the hexadecyltrimethylammonium 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; and the inactivation is carried out with formaldehyde solution.

4. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 1, characterized in that: The preparation method of the recombinant PVAX1-OmpA plasmid is: (I) Construction of recombinant PVAX1-OmpA plasmid vector: synthesize the outer membrane protein OmpA gene of Riemerella anatipestifer, connect the outer membrane protein OmpA gene with the eukaryotic expression vector PVAX1 through enzyme digestion to construct a recombinant PVAX1-OmpA plasmid, and transfer it into Escherichia coli DH5α; (ii) Large-scale preparation of recombinant PVAX1-OmpA plasmid: After the Escherichia coli DH5α is fermented and cultured, the bacteria are collected by centrifugation, and hydrochloric acid buffer, sodium hydroxide solution and acetic acid solution are added in sequence, and post-treatment is performed to obtain a purified recombinant PVAX1-OmpA plasmid solution; bacterial endotoxins are removed from the purified recombinant PVAX1-OmpA plasmid solution to obtain a large-scale prepared recombinant PVAX1-OmpA plasmid.

5. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 4, characterized in that: In step (ii), 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.

6. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 4, characterized in that: The formula of the hydrochloric acid buffer solution is 25 mM Tris-HCl, 10 mM EDTA, and 50 mM glucose; the formula of the sodium hydroxide solution is 250 mM NaOH and 1% SDS; and the formula of the acetic acid solution is 3 M potassium acetate and 5 M acetic acid.

7. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 1, characterized in that: 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.

8. The method for preparing a Riemerella anatipestifer composite vaccine according to claim 1, characterized in that: In parts by weight, the white oil for injection is 94 parts, aluminum stearate is 2 parts, Siben 80 is 6 parts, Tween 80 is 4 parts, the semi-finished product is 96 parts, the oil phase is 2 parts, and the water phase is 1 part.

9. A method for preparing Riemerella anatipestifer egg yolk antibody, characterized in that: The specific steps include: The composite vaccine prepared by the preparation method of the composite vaccine of Riemerella anatipestifer according to any one of claims 1 to 8 is used to immunize laying hens, and eggs are collected after immunization to obtain highly immune eggs, and the highly immune eggs are extracted to obtain Riemerella anatipestifer yolk antibodies.

10. The method for preparing Riemerella anatipestifer egg yolk antibody according to claim 9, characterized in that: The vaccine was administered three times, with an interval of 14 days between each immunization. The injection volume of the vaccine was 0.5 ml per animal.

Images