CN107670028B - Composite oil emulsion vaccine of riemerella anatipestifer inactivated vaccine and antibody and preparation method thereof - Google Patents
Composite oil emulsion vaccine of riemerella anatipestifer inactivated vaccine and antibody and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a composite oil emulsion adjuvant vaccine of a riemerella anatipestifer inactivated vaccine and an antibody and a preparation method thereof, the composite oil emulsion adjuvant vaccine is prepared by three times of emulsification technology of riemerella anatipestifer serum 1, a 2-type divalent inactivated vaccine, a mineral oil adjuvant and riemerella anatipestifer serum 1, 2-type divalent refined egg yolk antibody, and is a water-in-oil-in-water (W/O/W) emulsion, the finished product stability is good, the preparation operation is simple, and the injection and the inoculation are easy. The composite oil emulsion adjuvant vaccine realizes that the riemerella anatipestifer inactivated vaccine and the specific antibody thereof are mixed and do not meet, the immune effects are added and complemented without any adverse effect, the problem of blank period of immunity of the traditional inactivated vaccine is successfully solved, and the high-efficiency immune effect of effective immunity generated by inoculation is realized.
Description
Technical Field
The invention belongs to the technical field of vaccines, and particularly relates to a composite oil emulsion vaccine of a riemerella anatipestifer inactivated vaccine and an antibody and a preparation method thereof.
Background
The vaccine is characterized in that Rimerella Antipestifer (RA) is a pathogenic bacterium of infectious serositis of ducks and geese, the ducks and the geese with the age of 2-5 weeks are particularly easy to infect RA, the morbidity is 100%, the mortality is usually 30% -90%, the RA inactivated vaccine prepared by adding vaccine adjuvants such as aluminum hydroxide gel, propolis and mineral oil adjuvant has a good prevention effect on the infectious serositis of the ducks and the geese, 25 serotypes of RA are known, the inactivated vaccines prepared by different serotypes of RA lack cross immunity protection, the commercial production of the RA serum type 1 inactivated oil emulsion vaccine is approved in 2005, the commercial production of the RA serum type 1 and 2 type divalent oil emulsion vaccines is approved in 2010, the vaccine is efficient, the infectious serositis of the geese and the ducks should be well controlled theoretically, the actual situation of the geese and the duck breeding in the world is only widely reported from near ten years to the onset of various serositis of various documents in China, the RA, the current infectious serositis of the geese and the vaccine can be used for inducing the RA to vaccinate the vaccine for the 18-day-age of the ducks and the adult ducks, the vaccine is an effective vaccine for inducing the vaccine for the adult 4-day-age of the 4-age of the geese, even if the RA is found, the vaccine is an effective vaccine for the infectious serositis of the vaccine for the early-induced by using the early-4-year-age domestic duck, the early-age inactivated vaccine, the vaccine is an effective vaccine is found in the vaccine for the early-4-age-4-age vaccine, the early-4-year vaccine, the early-year vaccine is an infectious serositis vaccine is an infectious serological vaccine.
The serum of the RA-onset resistant duck contains protective antibodies capable of protecting ducklings against RA virulent attack, but the duck serum resistant to RA cannot realize commercial production. The RA inactivated vaccine is used for immunizing laying hens for multiple times, egg yolks of eggs of the laying hens contain high-titer antibodies, and after injection administration, ducklings also obtain resistance to RA virulent attack. Through determination, the immune protection period of the specific egg yolk antibody of the chicken for resisting RA on the susceptible ducklings can reach more than 10 days. A plurality of researches prove that the susceptible days age of ducks and geese to RA is 10-35 days age. Therefore, chicken anti-RA egg yolk antibody can not protect the whole immune of ducks and geese in the growing period.
Because RA inactivated vaccine and chicken anti-RA yolk antibody can not provide immune protection for ducks and geese in the whole process, antibiotics are widely applied to prevention of infectious serositis of ducks and geese, so that the antibiotics are widely used in breeding of the ducks and the geese, and the drug resistance problem and drug residue caused by the antibiotics become the food safety problem which is worried about by the government and the public in China at present and need to be solved urgently.
Disclosure of Invention
The technical problem is as follows:
1. after the injection of the riemerella anatipestifer egg yolk antibody solution, an organism quickly absorbs the riemerella anatipestifer egg yolk antibody solution into blood to form a specific passive immune effect, but the effect maintenance time is short and is 10-14 days. The riemerella anatipestifer inactivated oil emulsion vaccine induces ducks and geese to generate strong active immunity, and the efficacy maintaining time is 1-3 months. Unfortunately, the riemerella anatipestifer inactivated oil emulsion vaccine has an immune blank period of 10-14 days after the duck and goose are immunized, and the vaccine is infected and attacked before the immune efficacy is generated after 4-7 days of vaccination, so that immune failure occurs. The duck and the goose are highly susceptible to riemerella anatipestifer at the age of 10-35 days. Therefore, the riemerella anatipestifer inactivated oil emulsion vaccine and the hyperimmune yolk antibody thereof cannot protect ducks and geese from infectious serositis in the susceptible day age stage when being used independently.
2. The invention aims to solve the core technical problem that whether the immune protection effect of the riemerella anatipestifer inactivated oil emulsion vaccine and the antibody thereof can realize additive complementation? is the core technical problem to be solved by the invention.
3. The addition and complementation of the riemerella anatipestifer inactivated oil emulsion vaccine and the immune protection effect of the antibody are realized, the programmed release of the antibody and the vaccine is realized, namely the antibody quickly enters blood to play a specific passive immune protection effect, the efficacy maintenance time of the vaccine just fills the blank immune period required by the vaccine to induce specific active immunity, and the immune efficacy of the vaccine is not influenced. This program release technique is another key technique to be solved by the present invention.
In a word, the invention aims to provide a riemerella anatipestifer inactivated vaccine and a composite oil emulsion vaccine of the antibody and a preparation method thereof, and solves the technical problems that the riemerella anatipestifer antibody and the inactivated vaccine thereof are mixed after being isolated, the encounter reaction cannot occur, and the respective immune efficacy can be best played by programmed release, and the former cannot influence the latter; the immune efficacies of the riemerella anatipestifer inactivated vaccine and the antibody are added and complemented, so that an effective vaccine is provided for preventing and controlling the infectious serositis of the ducks and the geese, and the immunity and the survival rate of the ducks are effectively improved.
To achieve the above object of the invention, the following embodiments are adopted:
in one embodiment, the riemerella anatipestifer composite oil emulsion vaccine of the invention is composed of an immune prophase containing riemerella anatipestifer serum 1 and type 2 divalent inactivated bacterins, an oil phase containing a mineral oil adjuvant and an antibody phase containing riemerella anatipestifer serum 1 and type 2 divalent refined egg yolk antibodies, wherein the immune phase and the antibody phase are completely separated by the oil phase, the immune phase is in an inner layer, the oil phase is in an intermediate, and the antibody phase is in an outer peripheral layer, and is characterized in that:
the immunogen phase contains 2.0-4.0% w/v of emulsifier,
the oil phase contains 92-96% v/v white oil, 4-8% v/v emulsifier and 0.5-4% w/v stabilizer, or the oil phase is mineral oil adjuvant Montanide ISA 7VG,
the antibody phase contains 0.3-3.8% w/v emulsifier.
The composite oil emulsion vaccine is a water-in-oil-in-water emulsion, wherein the immune antigen phase accounts for 15.25-31.67% v/v, the oil phase accounts for 40.67-71.25% v/v, and the antibody phase accounts for 5.0-39.0% v/v.
In the complex oil emulsion vaccine of the present invention, the emulsifier in the immunogen phase and the emulsifier in the antibody phase are selected from any one of tween series with HLB value of 9.6-16.7, preferably tween 80. The emulsifier in the oil phase is selected from any one of Span (Span) or Arlacel with HLB value of 1.8-6.7, preferably Span (Span)80, or the oil phase comprises an oil phase adjuvant selected from one of Montanide ISA series, preferably MONTANIDE ISA 71 VG.
In another embodiment, the present invention provides a method for preparing a riemerella anatipestifer complex oil emulsion vaccine, comprising the steps of:
1) preparing the vaccine: inoculating Riemerella anatipestifer serum 1 and type 2 production strains to LB broth respectively at 37 ℃, performing shake culture at 180-220 r/min for 12-18 h to prepare seed solutions, performing amplification culture according to the seed solution addition ratio of 2-6%, and harvesting A525Bacterial liquid of 2.0 + -0.2 degreeAdding 37-40% formaldehyde solution to make the final concentration 0.15-0.3%, inactivating for 16-24 h at 37 ℃, and equivalently mixing the two serotype inactivated bacteria solutions to obtain the riemerella anatipestifer serum 1, 2 type bivalent inactivated vaccine;
2) preparation of immunogen phase: adding an emulsifier into the riemerella anatipestifer serum 1 and 2 type divalent inactivated vaccine solution obtained in the step 1) to enable the final concentration to be 2.0-4.0%, and uniformly mixing to obtain an immunogen phase;
3) preparation of oil phase: mixing white oil accounting for 92-96% v/v, emulsifier accounting for 4-8% v/v and stabilizer accounting for 0.5-4% (w/v), sterilizing and uniformly mixing to obtain an oil phase;
4) preparation of antibody phase: performing agar immunodiffusion test on antibody titer of 1: 16-64 (4log 2-6 log2) on riemerella anatipestifer serum 1 and 2 divalent refined egg yolk antibody, adding an emulsifier to enable the final concentration to be 0.3-3.8%, shaking and uniformly mixing to obtain an antibody phase;
5) preparation of a complex oil emulsion vaccine: according to the volume percentage, the immune antigen phase containing the riemerella anatipestifer serum 1 and the type 2 bivalent bacterin accounts for 15.25 to 31.67 percent, the oil phase accounts for 40.67 to 71.25 percent, the antibody phase containing the riemerella anatipestifer serum 1 and the type 2 refined egg yolk antibody accounts for 5.0 to 39.0 percent, and the three-effect secondary emulsification technology is carried out: and stirring and homogenizing the immunogen phase and the oil phase, and then adding the antibody phase to prepare water-in-oil-in-water emulsion to obtain the riemerella anatipestifer composite oil emulsion vaccine.
In another embodiment of the above method for preparing the riemerella anatipestifer composite oil emulsion vaccine, in step 1), the riemerella anatipestifer serum 1 and type 2 production strains are any strains which are separated from naturally-diseased ducks and geese and have good immunogenicity and meet the requirements of serotypes, and are preferably SC strains of serum 1 and AF strains of serum 2 separated from ducks; in the step 1), the expanded culture condition of the riemerella anatipestifer production strain is 220 r/min, and the culture is carried out for 16 h; the inactivation conditions of the liquid 1 and 2 of the riemerella anatipestifer serum are optimized to be 0.2 percent of final concentration of formaldehyde, the shaking rotating speed is 180 r/min, and the inactivation is carried out for 16 hours.
In another embodiment of the present invention, the preparation of riemerella anatipestifer is performedIn the steps 2) and 4), the emulsifier is any one of Tween series with the HLB value of 9.6-16.7 in the hydrophilic emulsifier, preferably Tween (Tween)80, and the final concentration of the Tween series in the immunogen phase is 2%. In the step 3), the mineral oil is selected from any one of refined white oil No. 10, imported white oil and vegetable oil, or the oil phase is MONTANIDETMISA 71 VG; the emulsifier is any one of span or Arlacel with a lipophilic emulsifier HLB value of 1.8-6.7, preferably span 80.
Preferably, in the method of the present invention, in step 3), the percentages of the components in the oil phase are: 94% v/v white oil, 6% v/v emulsifier, 2% w/v aluminium stearate.
Preferably, in the method of the present invention, in step 4), the riemerella anatipestifer serum 1 and 2 type divalent refined egg yolk antibody is any one of lipid-free and virus-free and bacterial microorganism-free refined egg yolk antibodies extracted from chicken, duck and goose eggs containing the riemerella anatipestifer serum 1 and 2 type high titer antibodies, and the riemerella anatipestifer serum 1 and 2 type divalent refined egg yolk antibody is any one of injection, freeze-drying and sterile powder; the agar immunodiffusion assay antibody titer was 1:32(5log 2).
Preferably, in the method of the present invention described above, in step 4), the final concentration of the emulsifier in the antibody phase is 0.3%. In the step 5), the volume percentages of the immunogen phase, the oil phase and the antibody phase in the water-in-oil-in-water emulsion are 20.33%, 40.67% and 39%; the third emulsification technology comprises the steps of first emulsification, adding an immunogen phase into an oil phase, wherein the rotating speed of a refiner is 6500r/min at the initial stage, the rotating speed of the refiner is 24000 r/min after the addition of the immunogen phase is finished, and the stirring time is 5 min; emulsifying for the second time, adding an antibody phase, and stirring for 30 seconds at the rotating speed of a homogenizer of 6500 r/min; emulsifying for the third time, and reversing the tank body frequency for 20-30 times/min for 3-5 min; the prepared composite oil emulsion adjuvant vaccine is a water-in-oil-in-water (w/o/w) milky emulsion, the conventional detection indexes of the vaccine comprise that the vaccine is dripped on the water surface and spreads in a cloudiness shape, 10mL of sample is centrifuged at 3000 r/min for 15min, and the amount of the lower layer clear liquid is less than or equal to 2.2mL, thus obtaining a qualified product.
In a specific embodiment, the method for preparing the riemerella anatipestifer composite oil emulsion vaccine comprises the following steps:
1) preparing the vaccine: taking Riemerella anatipestifer serum 1 and type 2 strains, respectively inoculating rabbit blood chocolate LB agar plates, culturing at 37 ℃ for 16 □ 24h, carrying out pure crushing inspection and serotype detection to be qualified, selecting a single colony, inoculating LB broth, and carrying out shaking culture at 180-220 r/min for 12-18 h to prepare seed liquid. Adding 2 percent □ 6 percent of seed liquid according to the proportion for carrying out amplification culture, and harvesting A525Adding a formaldehyde solution (the content is 37-40%) with the final concentration of 0.15-0.3% into the bacterial liquid when the bacterial liquid is 2.0 +/-0.2, sealing the bottle mouth, inactivating for 16h □ 24h by shaking at 37 ℃ (the rotating speed is 160-180 r/min), and performing sterile inspection to obtain the inactivated vaccine of the riemerella anatipestifer. Equivalently mixing the inactivated bacteria liquid of the type 1 and the type 2 of the riemerella anatipestifer serum to prepare divalent inactivated vaccine of the type 1 and the type 2 of the riemerella anatipestifer serum;
2) preparation of immunogen phase: adding an emulsifier into the riemerella anatipestifer serum 1 and 2 type divalent inactivated vaccine solution prepared in the step 1) to ensure that the final concentration is 2.0-4.0%, and shaking and uniformly mixing to obtain the immunogenic phase.
3) Preparation of oil phase: mixing refined white oil (No. 10) or imported white oil 92-96%, emulsifier 4-8% and stabilizer 0.5-4% (w/v), autoclaving at 121 deg.C for 20min, shaking, and mixing to obtain oil phase;
4) preparation of antibody phase: the method comprises the following steps of (1) determining the agar immunodiffusion test antibody titer to be 1: 16-64 (4log 2-6 log2) by using riemerella anatipestifer serum 1 and 2 antigens, adding an emulsifier to enable the final concentration to be 0.3% -3.8%, shaking and uniformly mixing to obtain an antibody phase;
5) preparation of a complex oil emulsion adjuvant vaccine: according to the volume, in the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody, an immune original phase prepared from the riemerella anatipestifer serum 1 and the 2-type divalent inactivated vaccine accounts for 15.25-31.67%, an oil phase prepared from a mineral oil adjuvant accounts for 40.67-71.25%, and an antibody phase prepared from the riemerella anatipestifer serum 1 and the 2-type refined egg yolk antibody accounts for 5.0-39.0%. And (3) adopting a three-time emulsification technology to process the immune original phase, the oil phase and the antibody phase into a water-in-oil-in-water (w/o/w) emulsion to obtain the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody.
In the above specific embodiment, in step 1), the riemerella anatipestifer serum 1 and type 2 strains are any strains which are separated from naturally diseased ducks and geese and have good immunogenicity and meet the requirements of serotypes, and are preferably SC strains of serum 1 and AF strains of serum 2 separated from killed ducks; the preferred condition of the expanded culture of the riemerella anatipestifer strain is that the shaking culture rotating speed is 220 r/min and the time is 16 h; the inactivation conditions of the bacterial liquid of the 1 and 2 types of the riemerella anatipestifer serum are optimized to be 0.2 percent of the final concentration of formaldehyde, the shaking rotating speed is 180 r/min, and the inactivation time is 16 h.
In the above specific embodiment, in the method of the present invention, steps 2) and 4), the hydrophilic emulsifier is any one of Tween (Tween) series having HLB value of 9.6-16.7, preferably Tween (Tween)80, and the final concentration thereof in the immunogen phase is optimized to 2%.
In the above specific embodiment, in the step 3), the mineral oil is any one of refined white oil (No. 10) and imported white oil, and further comprises vegetable oil, and the oil phase is an oil phase prepared from mineral oil and vegetable oil, and is specifically selected from commercial oil adjuvant ONTANIDETMAny of the ISA families, such as: MONTANIDETMISA 71 VG; the lipophilic emulsifier is Span (Span) or Arlacel with the HLB value of 1.8-6.7, is optimized to Span (Span)80, and preferably, the mixing ratio of the components of the oil phase is preferably refined white oil (No. 10) or imported white oil 94% (v/v), emulsifier 6% (v/v) and aluminum stearate 2% (w/v).
In the above embodiment, in the method of the present invention, in step 4), the riemerella anatipestifer serum 1 and 2 type bivalent refined egg yolk antibody is any one of lipid-free and refined egg yolk antibodies free of microorganisms such as viruses and bacteria extracted from chicken, duck and goose eggs containing the riemerella anatipestifer serum 1 and 2 type high titer antibodies; the form of the riemerella anatipestifer serum 1 and 2 type bivalent refined egg yolk antibody is any one of injection, freeze-drying agent and sterile powder injection.
In the above embodiment, the method of the present invention, step 4), in the method for detecting antibody titer of divalent refined egg yolk type 1, 2 of riemerella anatipestifer serum, the detection antigen used in the method is any one of antigens prepared by an ultrasonic method, a high pressure lysis method and a thermal phenol method, preferably a polysaccharide antigen prepared by a thermal phenol method, and the agar immunodiffusion test antibody titer is 1:32(5log 2); the final concentration of emulsifier in the antibody phase was optimized to 0.3%.
In the above embodiment, the method of the present invention, step 5), the mixing ratio of the immune antigen phase prepared from the riemerella anatipestifer serum 1, the divalent inactivated vaccine type 2, the oil phase of the mineral oil emulsion adjuvant and the antibody phase prepared from the riemerella anatipestifer serum 1, the divalent refined egg yolk antibody type 2 is optimized to be 20.33%, 40.67% and 39% (v/v); the three-time emulsification condition is optimized as the first emulsification, the rotating speed of a homogenizer is 6500r/min at the initial stage, the rotating speed is adjusted to be more than 24000 r/min after the addition of the immunogen is finished, and the stirring time is 5 min; emulsifying for the second time, wherein the rotating speed of a homogenizer is 6500r/min, and stirring for 30 seconds; emulsifying for the third time, and inverting the tank body, wherein the frequency is 20-30 times/min, and the time is 3-5 min; the obtained compound oil emulsion adjuvant vaccine is water-in-oil-in-water (w/o/w) milky emulsion, the conventional detection indexes comprise that the vaccine is dripped on the water surface and spreads in a cloudiness shape, and after 10mL of sample is centrifuged for 15min at 3000 r/min, the lower layer clear liquid is less than or equal to 2.2mL, thus obtaining a qualified product.
In the above specific embodiment, in the method of the present invention, in step 5), the finished product of the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody can be further classified into any one of a stable emulsion and a short-term stable emulsion; one of the finished products of the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody is a stable emulsion, namely the stable emulsion does not have a layering phenomenon when being stored at 4-8 ℃ or room temperature, does not need to shake and mix uniformly when being injected and inoculated to animals, the other one of the finished products is a short-term stable emulsion, namely the stable emulsion is a milky emulsion which appears a layering phenomenon after being placed at 4-8 ℃ or room temperature for 3h, and the lower layer is a turbid and opaque liquid which gradually becomes a clear and transparent aqueous solution. The product is in uniform milky emulsion state within 3h after shaking for 1min, has no demixing phenomenon, and can ensure uniformity of the product during injection and inoculation of animals. In other words, the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody has to be shaken and mixed evenly for the clinical use.
In the above embodiments, the finished product of the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer inactivated vaccine and the antibody in the method of the present invention, step 5) can be further classified into any one of stable emulsion and short-term stable emulsion. The short-term stable emulsion of the finished product is divided into an upper layer and a lower layer after being stored at 4-8 ℃ or room temperature, wherein the upper layer is milky emulsion, and the lower layer is turbid and opaque and gradually becomes clear and transparent aqueous solution. After shaking for 1min, the emulsion is uniform milky emulsion within 3 h. Therefore, the finished product must be shaken and mixed evenly when used.
Drawings
FIG. 1: a process flow schematic diagram of a composite oil emulsion vaccine manufacturing process of the riemerella anatipestifer inactivated vaccine and the antibody;
FIG. 2: the vaccine has two physical appearance traits of a finished product of the riemerella anatipestifer inactivated vaccine and a finished product of the compound oil emulsion adjuvant vaccine of the antibody.
Detailed Description
The following examples are presented to further illustrate and understand the spirit of the present invention, but are not intended to limit the scope of the present invention in any way.
Example 1 preparation of Riemerella anatipestifer Complex emulsion vaccine
(the preparation process is shown in figure 1)
1) Preparing the vaccine: producing strains from a riemerella anatipestifer serum 1 type SC strain and a serum 2 type AF strain, respectively inoculating the strains to a rabbit blood chocolate LB plate, culturing for 24-36 h at 37 ℃, selecting a single colony to inoculate in 5mL LB broth after passing a pure-crushing inspection, and performing shaking culture for 16h at 37 ℃ at 220 r/min, wherein the seeds are seed liquid after passing the pure-crushing inspection. Adding seed liquid in a proportion of 4%, performing 500mL amplification culture, and waiting for bacterial liquidA525When the concentration is 2.0 +/-0.2 percent, adding formaldehyde solution (the content is 37 to 40 percent) with the final concentration of 0.2 percent, and sealing the bottle mouth. Placing the mixture at a rotating speed of 180/min, oscillating and inactivating the mixture at 37 ℃ for 16h, performing sterile inspection to be qualified, and mixing the SC strain inactivated bacteria liquid and the AF strain inactivated bacteria liquid in equal amount to obtain the Riemerella anatipestifer serum 1 and 2 type bivalent inactivated bacterin.
2) Preparation of immunogen phase: sterile Tween (Tween)80 was added to the inactivated vaccine obtained in the above 1) to give a final concentration of 2.0% (volume concentration v/v). Shaking and mixing to obtain the immune antigen.
3) Preparation of oil phase: adding 470mL of imported white oil, 8030mL of span and 10g of aluminum stearate into a 500mL saline bottle, sterilizing at 121 ℃ under high pressure for 20min, and shaking and mixing uniformly to obtain an oil phase;
4) preparation of antibody phase: extracting the riemerella anatipestifer serum 1 and 2 type bivalent refined egg yolk antibody by adopting an octanoic acid method, detecting the asepsis and the virus to be qualified, determining the agar immunodiffusion test antibody titer of the riemerella anatipestifer serum 1 and 2 type antigen prepared by a hot phenol method to be 1:16(4log2), adding sterile Tween (Tween)80 to ensure that the final concentration is 0.3% (v/v), shaking and mixing uniformly to obtain the antibody phase.
5) Preparation of a complex oil emulsion vaccine: according to the volume percentage, the matching proportion of the immune antigen prepared from the riemerella anatipestifer serum 1 and the divalent vaccine type 2, the oil phase prepared from the mineral oil adjuvant and the antibody prepared from the riemerella anatipestifer serum 1 and the divalent refined yolk antibody type 2 is 20.33%, 40.67% and 39%. According to the aseptic operation, firstly adding 120mL of oil phase into an emulsification tank of a homogenizer, regulating the rotating speed to 6500r/min, slowly adding 60mL of immune original phase while stirring, increasing the rotating speed to 21500r/min after the addition is finished, emulsifying for 3min, and stopping the machine, wherein the first emulsification is realized. 61mL of the first emulsion product was added to another sterile emulsion tank and 39mL of the antibody phase was added. Starting the homogenizer, regulating the rotating speed to 6500r/min, obtaining the emulsion with the emulsifying time of 30 seconds, and stopping the machine, wherein the second emulsification is realized. The emulsion was poured into a 100mL sterile saline bottle and the mouth was sealed with a reverse-stoppered stopper. After standing for 1h, the bottles are inverted and mixed evenly for 3min, with the frequency of about 20 to 30 times/min. Sampling, inspecting, and obtaining the qualified composite oil emulsion vaccine containing the riemerella anatipestifer serum 1, the 2-type divalent inactivated vaccine and the antibody, which is shown in figure 2.
6) And (3) performing immune challenge test on the composite oil emulsion vaccine of the riemerella anatipestifer inactivated vaccine and the antibody obtained in the previous step. 10-day-old non-immunized ducks were divided into 8 groups of 11 ducks. The injection dosage of the composite oil emulsion adjuvant vaccine of the riemerella anatipestifer serum 1 and the divalent inactivated vaccine of the riemerella anatipestifer type 2 and the antibody is 0.5 mL/vaccine, the injection dosage of the riemerella anatipestifer serum 1 and the divalent inactivated oil emulsion adjuvant vaccine of the riemerella anatipestifer type 2 is 0.3 mL/vaccine, the injection dosage of the riemerella anatipestifer serum 1 and the divalent refined yolk antibody of the riemerella anatipestifer type 2 is 0.2 mL/vaccine, the vaccine or the antibody is injected in a leg muscle, and the virus counteracting dosage is 200LD 50. The observation was continued for 7 days after challenge. The results are shown in Table 1.
7) And 5) the immunity efficacy of the composite oil emulsion vaccine of the riemerella anatipestifer inactivated vaccine and the antibody is obtained by adopting homologous strains for virus attack, and the test results are detailed in table 2.
As can be seen from the data in Table 1, the Relative Protection Rate (RPR) counted by the morbidity 7 days after the immunization of the composite oil emulsion vaccine of the riemerella anatipestifer inactivated vaccine and the antibody reaches 81.8 percent, and is increased by 27.3 percent compared with 54.5 percent of the traditional riemerella anatipestifer inactivated oil emulsion vaccine group. 18 days after immunization, the relative protection rate of the riemerella anatipestifer inactivated vaccine group and the compound oil emulsion vaccine group of the antibody and the traditional riemerella anatipestifer inactivated oil emulsion vaccine group is 100 percent. And the relative protection rate of the single riemerella anatipestifer serum 1 and 2 type bivalent refined yolk antibody group is 36.4 percent. The result shows that the antibody in the composite oil emulsion vaccine of the riemerella anatipestifer inactivated vaccine and the antibody naturally disappears, and the active immune efficacy of the vaccine is not influenced. Table 2 shows the result of challenge test for riemerella anatipestifer serotype 2 homologous strain, showing similar effect as above.
TABLE 1 composite oil emulsion adjuvant vaccine of inactivated vaccine and antibody against Riemerella anatipestifer for duck immunization and challenge test result with homologous strain of serum type 1
Number of episodes or deaths/number of vaccinated animals,
relative protection calculated for morbidity/relative protection calculated for mortality. RPR (abbreviation for Relative protection rate), the Relative protection rate, was calculated as (control morbidity or mortality-test morbidity or mortality)/control morbidity or mortality 100. TABLE 2 Duck Riemerella inactivated vaccine and antibody composite oil emulsion adjuvant vaccine for duck immunization and then challenge results with serum type 2 homologous strain
Number of episodes or deaths/number of vaccinated animals,
relative protection calculated for morbidity/relative protection calculated for mortality. RPR (abbreviation for Relative protection rate), the Relative protection rate, was calculated as (control morbidity or mortality-test morbidity or mortality)/control morbidity or mortality 100.
The present invention is not limited to the above embodiments, and all the strains and formulas of the same or other serotypes as those of the above embodiments of the present invention are within the protection scope of the present invention without departing from the spirit and scope of the present invention.
Claims (23)
1. A riemerella anatipestifer composite oil emulsion vaccine comprises an immune original phase containing riemerella anatipestifer serum 1 and type 2 divalent inactivated vaccine, an oil phase containing a mineral oil adjuvant and an antibody phase containing riemerella anatipestifer serum 1 and type 2 divalent refined egg yolk antibody, wherein the immune phase and the antibody phase are completely isolated by the oil phase, the immune phase is in an inner layer, the oil phase is in an intermediate, and the antibody phase is in a peripheral layer, and the riemerella anatipestifer composite oil emulsion vaccine is characterized in that: the emulsion is an aqueous-in-oil-in-aqueous system, wherein an immunogen phase accounts for 15.25-31.67% v/v, an oil phase accounts for 40.67-71.25% v/v, and an antibody phase accounts for 5.0-39.0% v/v;
wherein the immunogen phase contains 2.0-4.0% w/v of emulsifier, the oil phase contains 92-96% v/v of white oil, 4-8% v/v of emulsifier and 0.5-4% w/v of stabilizer, or the oil phase is commercial mineral oil adjuvant MONTANIDE TMISA 71VG, and the antibody phase contains 0.3-3.8% w/v of emulsifier.
2. The complex oil emulsion vaccine of claim 1, wherein the emulsifier in the immunogen phase and the emulsifier in the antibody phase are selected from any one of tween series with HLB value of 9.6-16.7.
3. The complex oil emulsion vaccine of claim 2, wherein the emulsifier in the immunogen phase and the antibody phase is tween 80.
4. The complex oil emulsion vaccine of claim 1, wherein the emulsifier in the oil phase is selected from any one of span or Arlacel with HLB value of 1.8-6.7.
5. The complex oil emulsion vaccine of claim 1, wherein the emulsifier in the oil phase is Span 80.
6. The complex oil emulsion vaccine of claim 1 wherein the oil phase is MONTANIDE ISA 71 VG.
7. A method for preparing a riemerella anatipestifer composite oil emulsion vaccine comprises the following steps:
1) preparing the vaccine: inoculating Riemerella anatipestifer serum 1 and type 2 production strains to LB broth respectively at 37 ℃, performing shake culture at 180-220 r/min for 12-18 h to prepare seed solutions, performing amplification culture according to the seed solution addition ratio of 2-6%, and harvesting A525Adding a formaldehyde solution with the content of 37-40% into the bacterial liquid with the bacterial liquid content of 2.0 +/-0.2 to ensure that the final concentration is 0.15-0.3%, inactivating the bacterial liquid at 37 ℃ for 16-24 h, and equivalently mixing the two serotype inactivated bacterial liquids to obtain the riemerella anatipestifer serum 1 and 2 type bivalent inactivated vaccine;
2) preparation of immunogen phase: adding an emulsifier into the riemerella anatipestifer serum 1 and 2 type divalent inactivated vaccine solution obtained in the step 1) to enable the final concentration to be 2.0-4.0% v/v, and uniformly mixing to obtain an immunogen phase;
3) preparation of oil phase: mixing white oil accounting for 92-96% v/v, emulsifier accounting for 4-8% v/v and stabilizer accounting for 0.5-4% w/v, sterilizing and uniformly mixing to obtain an oil phase;
4) preparation of antibody phase: performing agar immunodiffusion test on a 1: 16-64 antibody titer of riemerella anatipestifer serum type 1 and type 2 bivalent refined egg yolk antibodies, adding an emulsifier to enable the final concentration to be 0.3-3.8%, shaking and uniformly mixing to obtain an antibody phase;
5) preparation of a complex oil emulsion vaccine: according to the volume percentage, the immune antigen phase containing the riemerella anatipestifer serum 1 and the type 2 bivalent bacterin accounts for 15.25-31.67%, the oil phase accounts for 40.67-71.25%, the antibody phase containing the riemerella anatipestifer serum 1 and the type 2 refined egg yolk antibody accounts for 5.0-39.0%, and the three-time emulsification technology is carried out: and stirring and homogenizing the immunogen phase and the oil phase, and then adding the antibody phase to prepare water-in-oil-in-water emulsion to obtain the riemerella anatipestifer composite oil emulsion vaccine.
8. The method according to claim 7, wherein in the step 1), the riemerella anatipestifer serum 1 and type 2 production strain is any strain which is separated from naturally-occurring ducks and geese and meets the serotype requirement and has immunogenicity.
9. The method according to claim 8, wherein in the step 1), the riemerella anatipestifer serum 1, type 2 production strains are serum type 1 SC strains and serum type 2 AF strains separated from ducks.
10. The method of claim 7, wherein in the step 1), the expanded culture condition of the riemerella anatipestifer producing strain is 220 r/min, and the culture is carried out for 16 h.
11. The method according to claim 7, wherein in the step 1), the bacterial liquid inactivation conditions of the riemerella anatipestifer serum 1 and the riemerella anatipestifer serum 2 are optimized to be 0.2% of final concentration of formaldehyde, the shaking rotation speed is 180 r/min, and the inactivation time is 16 h.
12. The method of claim 7, steps 2) and 4), wherein the emulsifier is tween 80, at a final concentration of 2% in the immunogenic phase.
13. The method according to claim 7, wherein in step 3), the white oil is selected from any one of refined white oil No. 10 and imported white oil.
14. The method according to claim 7, wherein in step 3), the oil phase is a commercial oil adjuvant MONTANIDE TMISA 71 VG.
15. The method of claim 7, step 3) wherein the emulsifier is span 80.
16. The method of claim 7, wherein in the step 3), the percentage of each component in the oil phase is as follows: 94% v/v white oil, 6% v/v emulsifier, 2% w/v aluminium stearate.
17. The method according to claim 7, wherein in the step 4), the bivalent refined yolk antibody of the riemerella anatipestifer serum 1 and 2 is any one of lipid-free and refined yolk antibody of virus-free and bacterial microorganisms extracted from chicken, duck and goose eggs containing the high titer antibody of the riemerella anatipestifer serum 1 and 2.
18. The method according to claim 7, wherein in the step 4), the riemerella anatipestifer serum 1 and 2 type bivalent refined egg yolk antibody is any one of injection, freeze-drying and sterile powder.
19. The method of claim 7, wherein in step 4) the agar immunodiffusion test antibody titer is 1: 32.
20. The method of claim 7, wherein in step 4), the final concentration of emulsifier in the antibody phase is 0.3%.
21. The method according to claim 7, wherein in step 5), the volume percentages of the immunogenic, oil and antibody phases in the water-in-oil-in-water emulsion are 20.33%, 40.67% and 39%.
22. The method according to claim 7, wherein in step 5), the three-time emulsification technique comprises the first emulsification, adding the immunogen phase into the oil phase, wherein the rotation speed of the homogenizer is 6500r/min in the initial stage, the addition of the immunogen phase is completed, the rotation speed of the homogenizer is 24000 r/min, and the stirring time is 5 min; emulsifying for the second time, adding an antibody phase, and stirring for 30 seconds at the rotating speed of a homogenizer of 6500 r/min; and emulsifying for the third time, namely inverting the tank body for 20-30 times/min for 3-5 min.
23. The method of claim 7, wherein in step 5), the complex oil emulsion adjuvant vaccine is a water-in-oil-in-water milky emulsion, and the conventional detection index comprises that the complex oil emulsion adjuvant vaccine is dispersed in a cloud form when being dropped on the water surface, and the lower layer clear liquid amount is less than or equal to 2.2mL after 10mL of sample is centrifuged for 15min at 3000 r/min.
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