CN108904796B

CN108904796B - Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof

Info

Publication number: CN108904796B
Application number: CN201810763400.6A
Authority: CN
Inventors: 范志宇; 王芳; 胡波; 魏后军; 宋艳华; 仇汝龙; 陈萌萌; 朱伟峰; 薛家宾
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Academy of Agricultural Sciences
Priority date: 2018-07-12
Filing date: 2018-07-12
Publication date: 2022-06-03
Anticipated expiration: 2038-07-12
Also published as: CN108904796A

Abstract

The invention relates to a rabbit hemorrhagic disease virus baculovirus vector, a pasteurella multocida disease bivalent inactivated vaccine and a preparation method thereof, belonging to the technical field of immunity. Inoculating recombinant rabbit hemorrhagic disease virus VP60 baculovirus to Sf9 insect cells, culturing at 27-28 ℃, harvesting cell cultures and inactivating when cytopathic effect reaches more than 85%, and taking the inactivated products as rabbit hemorrhagic disease virus antigens; performing amplification culture on a rabbit-derived capsular group A pasteurella multocida C51-17 strain, and inactivating a bacterial liquid to obtain an inactivated bacterial liquid as a pasteurella multocida antigen; the two antigens are mixed with an adjuvant according to a proportion to prepare the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida disease bivalent inactivated vaccine. The invention can provide a rabbit hemorrhagic disease virus baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine which have high safety, good immune effect and simple and convenient process, and are used for preventing and controlling rabbit viral hemorrhagic disease (rabbit plague) and rabbit pasteurella multocida disease.

Description

Rabbit hemorrhagic disease baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof

Technical Field

The invention relates to a rabbit hemorrhagic disease virus baculovirus vector, a pasteurella multocida disease bigeminal inactivated vaccine and a preparation method thereof, belonging to the technical field of immunity.

Background

Rabbit Hemorrhagic Disease (RHD) is an acute, virulent, and highly contagious disease caused by Rabbit Hemorrhagic Disease Virus (RHDV), commonly known as "Rabbit blast". The disease is often fulminant epidemic, the morbidity and the fatality rate are extremely high, and the mortality rate is about 90 percent. Rabbit Pasteurellosis (Pasteurellosis), also known as rabbit Hemorrhagic Septicemia (HS), is an acute, septic infectious disease caused by rabbit Pasteurella multocida (Pm). The disease can be divided into rhinitis type, pneumonia type, otitis media type and other types of diseases, and the pathological changes of various types of diseases are inconsistent, but two or more types of diseases are often combined. Rabbit hemorrhagic disease virus and pasteurella are listed as two types of pathogenic microorganisms and three types of pathogenic microorganisms respectively in China.

Rabbit hemorrhagic disease and pasteurella multocida disease are main epidemic diseases seriously endangering the healthy breeding of rabbits, and the current prevention and control measures are mainly vaccine immunity. Although the 'rabbit hemorrhagic disease and pasteurella multocida disease combined inactivated vaccine' exists in China, the antigen components of the vaccine are rabbit hemorrhagic disease tissue virus and pasteurella multocida. However, the vaccine has important defects, which are mainly shown in that the animal individual difference for preparing the rabbit hemorrhagic disease virus is large, the large-scale production of the vaccine antigen is not facilitated, the quality control is difficult to carry out, and most importantly, the preparation process of the vaccine is easy to cause the strong virus diffusion and the biological safety is poor. In addition, the preparation process of the pasteurella multocida is not stable enough, the difference of antigen immunogenicity among batches is large, and the toxic attack protection rate in the efficacy test is low. The defects seriously affect the quality of the vaccine and restrict the production efficiency of enterprises.

The large-scale suspension culture and the high-density fermentation culture of bacteria can realize the large-scale production of the vaccine, thereby being beneficial to the quality control of the vaccine, reducing the cost and simplifying the production process. The method is based on a new national veterinary drug 'rabbit hemorrhagic disease virus baculovirus vector inactivated vaccine (BAC-VP60 strain'), and by technical improvement, the production process of the vaccine antigen is improved, the immune efficacy of the original vaccine is enhanced, a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine are developed, the technical bottleneck of the traditional vaccine is broken through, the production and the quality stability of the vaccine are facilitated, and the updating of the vaccine is accelerated.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: the rabbit hemorrhagic disease baculovirus vector, the pasteurella multocida disease bivalent inactivated vaccine and the preparation method thereof are provided, and the method is easier for large-scale production, controllable in quality, stable in preparation process and good in biological safety. Compared with the existing vaccine, the vaccine has substantial improvement on the aspects of safety, immune challenge protection rate, immune period, storage life and the like.

In order to solve the technical problems, the invention provides a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine.

Further, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and a pasteurella multocida disease combined inactivated vaccine, wherein the antigen components in the combined inactivated vaccine are recombinant rabbit hemorrhagic disease virus VP60 baculovirus and pasteurella multocida.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease combined inactivated vaccine, wherein the antigen components in the combined inactivated vaccine are a recombinant rabbit hemorrhagic disease virus VP60 baculovirus vector cell culture and pasteurella multocida.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine, wherein the antigen component of the bivalent inactivated vaccine is a recombinant rabbit hemorrhagic disease VP60 baculovirus vector full-suspension culture cell culture and pasteurella multocida.

Furthermore, the invention provides a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine, wherein the antigen components in the bivalent inactivated vaccine are a recombinant rabbit hemorrhagic disease VP60 baculovirus vector cell culture and a pasteurella multocida zymocyte liquid.

Furthermore, the invention provides a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine, wherein the antigen components in the bivalent inactivated vaccine are a recombinant rabbit hemorrhagic disease VP60 baculovirus vector full-suspension culture cell culture and a pasteurella multocida zymocyte liquid.

Furthermore, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine, wherein the bivalent vaccine is prepared by inactivating a recombinant rabbit hemorrhagic disease virus VP60 baculovirus cell culture and pasteurella multocida zymocyte liquid.

Further, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and a pasteurella multocida disease combined inactivated vaccine, wherein the recombinant rabbit hemorrhagic disease virus VP60 baculovirus cell culture is harvested when lesion cells reach more than about 85%.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bigeminal inactivated vaccine, wherein pasteurella multocida zymocyte liquid adopts pasteurella multocida C51-17 strain freeze-dried bacteria as strains.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bigeminal inactivated vaccine, wherein pasteurella multocida zymocyte liquid adopts pasteurella multocida C51-17 strain freeze-dried bacteria subjected to solid culture as first-grade seeds.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the primary seeds are stored at the temperature of 2-8 ℃ and the service life is not more than 7 days.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bigeminal inactivated vaccine, wherein pasteurella multocida zymocyte liquid adopts pasteurella multocida C51-17 strain freeze-dried bacteria subjected to solid culture and liquid culture as secondary seeds.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the secondary seeds are stored at the temperature of 2-8 ℃ and the service life is not more than 4 days.

Further, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease combined inactivated vaccine, and the pure test is carried out on the secondary seeds.

Further, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine, wherein the pasteurella multocida zymocyte liquid is a zymocyte liquid cultured in a ventilating way.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine, wherein the pasteurella multocida zymocyte liquid is subjected to pure inspection after fermentation culture.

Furthermore, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the antigen is matched according to the fact that each milliliter of the vaccine contains VP60 protein before inactivation with the hemagglutination titer not less than 1: 256.

Furthermore, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the antigen contains not less than 7.5 multiplied by 10 pasteurella multocida bacteria number before inactivation per milliliter of vaccine⁹And (5) carrying out seedling matching according to the CFU standard.

Furthermore, the invention provides a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease combined inactivated vaccine, the antigen has the VP60 protein hemagglutination titer before inactivation of not less than 1:256 and the pasteurella multocida bacterium number in each milliliter of vaccineNot less than 7.5X 10⁹And (5) performing seedling matching according to the CFU standard.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the rabbit hemorrhagic disease baculovirus vector cell culture inactivated liquid is stored at the temperature of 2-8 ℃ for no more than 6 months.

Further, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, wherein the pasteurella multocida inactivated liquid is stored at the temperature of 2-8 ℃ for no more than 6 months.

Furthermore, the invention provides a combined inactivated vaccine of the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida disease, and the inactivated liquid of the cell culture of the rabbit hemorrhagic disease baculovirus vector is subjected to inactivation test before the vaccine is prepared.

Furthermore, the invention provides a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine, and inactivation test is carried out on pasteurella multocida inactivated liquid before the vaccine preparation.

Preferably, the rabbit hemorrhagic disease virus baculovirus vector is a recombinant rabbit hemorrhagic disease virus VP60 baculovirus BAC-VP60 strain, which is preserved in China center for type culture Collection in 6 months and 26 days in 2018, and the address is Wuhan university, Wuhan, the zip code 430072, and the preservation number is CCTCC V201833; the Pasteurella multocida is strain C51-17 (Pasteurella multocida C51-17), which is deposited in China center for type culture Collection in 26.6.2018, with the address of Wuhan, Wuhan university, zip code 430072, with the deposit number: CCTCC M2018401.

Preferably, the bivalent inactivated vaccine contains an adjuvant.

Preferably, the adjuvant is aluminum hydroxide gel, and the ratio of the antigen to the aluminum hydroxide gel is 9: 1.

The invention provides a preparation method of a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine, which comprises the following steps:

(1) preparation of rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid: inoculating recombinant rabbit hemorrhagic disease virus VP60 baculovirus into cells for culturing, and inactivating the harvested cell culture;

(2) preparation of pasteurella multocida inactivated solution: carrying out fermentation tank ventilation culture on the pasteurella multocida, and inactivating the zymocyte liquid;

(3) preparing a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine: the antigen content is not less than 1:256 according to the hemagglutination titer of the inactivated VP60 protein contained in each milliliter of vaccine, and the number of pasteurella multocida is not less than 7.5 multiplied by 10⁹And (5) preparing the vaccine according to the CFU standard, adding an adjuvant, and fully and uniformly mixing.

Further, the recombinant rabbit hemorrhagic disease virus VP60 baculovirus is subjected to suspension culture.

Further, the inoculated cells in the step (1) of the preparation method of the bivalent vaccine are Sf9 cells, the used culture medium is an insect cell culture medium, the inoculation amount of the recombinant rabbit hemorrhagic disease virus VP60 baculovirus is 1% of the total amount of the culture medium, the culture conditions are that the temperature is 27-28 ℃, the pH value is 6.0-6.2, the Dissolved Oxygen (DO) is 50-60%, and the stirring speed is 50-70 r/min; when the diseased cells reach more than 85 percent, the cell culture can be harvested.

Further, the rotating speed of the Sf9 cells during culture is the same as or different from the culturing speed of the Sf9 cells after inoculation of the recombinant rabbit hemorrhagic disease virus VP60 baculovirus.

Further, cells reached 2X 10 in Sf9⁶The recombinant rabbit hemorrhagic disease virus VP60 baculovirus is inoculated at about one/ml.

Further, cell cultures were harvested when diseased cells reached more than 90%.

Further, cell cultures were harvested when the diseased cells reached more than 95%.

Furthermore, after the cell culture of the baculovirus vector of the rabbit hemorrhagic disease virus is obtained in the step (1), the hemagglutination titer is required to be determined, and the hemagglutination titer of the cell culture to 1% human O-type red blood cells is not less than 1: 512.

Further, in the step (1), the harvested cell culture is subjected to inactivation treatment by using formaldehyde; and performing inactivation test on the inactivated rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation solution: after the inactivation solution is inoculated to the cells, the cells are free from cytopathic effect, and hemagglutination is measured by erythrocyte agglutination.

Further, in the step (1), the harvested cell culture is subjected to inactivation treatment by using a formaldehyde solution with a final concentration of 0.2%.

Further, in the step (1), the harvested cell culture is subjected to inactivation treatment using a formaldehyde solution while stirring.

Further, in the step (1), the agitation speed for inactivating the harvested cell culture by using the formaldehyde solution is the same as or different from the agitation speed for culturing the recombinant rabbit hemorrhagic disease virus VP60 baculovirus.

Further, in the step (1), the culture temperature for inactivating the harvested cell culture by using the formaldehyde solution is different from the culture temperature for culturing the recombinant rabbit hemorrhagic disease virus VP60 baculovirus.

Further, in the step (1), the culture temperature of the harvested cell culture subjected to inactivation treatment by using the formaldehyde solution is higher than that of the recombinant rabbit hemorrhagic disease virus VP60 baculovirus.

Further, in the step (1), inactivation test is performed on the inactivated recombinant rabbit hemorrhagic disease virus VP60 baculovirus vector cell culture.

Further, in the step (1), the culture temperature of the cell culture in the inactivation test is the same as that of the recombinant rabbit hemorrhagic disease virus VP60 baculovirus.

Further, in the step (2), the fermentation tank aeration culture is carried out on the pasteurella multocida, and the specific steps are as follows: inoculating a single colony of pasteurella multocida to a martin agar inclined plane containing serum and lysed whole blood, and culturing to obtain a first-grade pasteurella multocida seed; inoculating the primary seeds into a Martin broth containing serum, and culturing to obtain secondary pasteurella multocida seeds; according to the volume of the fermentation tank, 60-70% of fermentation medium and 0.01-0.02% of defoaming agent are filled, and the medium is sterilizedInoculating secondary seeds according to 5-10% of the amount of the culture medium, adding serum, stirring at a rotating speed of 200-300 r/min and a pH value of 7.2-7.6, ventilating and culturing, and harvesting a culture solution; the number of viable bacteria in each ml of culture solution should not be less than 1.2 × 10¹⁰CFU。

Further, in the step (2), the freeze-dried strain of pasteurella multocida C51-17 is subjected to fermenter aeration culture.

Further, in the step (2), the first-class seeds are stored at the temperature of 2-8 ℃ and the service life is not more than 7 days.

Further, in the step (2), the secondary seeds are stored at the temperature of 2-8 ℃ and the service life is not more than 4 days.

Further, in the step (2), a pure inspection is performed on the secondary seed.

Further, the obtained culture bacterial liquid is subjected to inactivation treatment by formaldehyde in the step (2), inactivation inspection is carried out, and after inactivation, a sample is taken and inoculated with Martin agar containing 4% of serum and 0.1% of lysed whole blood and a Pasteurella multocida fermentation culture medium for culture, and the culture is required to grow aseptically.

Further, the harvested culture broth is inactivated by 0.4% formaldehyde in the step (2).

Further, the culture temperature of the obtained culture bacterial liquid inactivated by formaldehyde in the step (2) is the same as the culture temperature of the pasteurella multocida fermentation culture.

Further, the stirring speed of the obtained culture bacterium liquid subjected to inactivation treatment by using formaldehyde in the step (2) is different from the stirring speed of the pasteurella multocida fermentation culture.

Further, the stirring speed of the obtained culture bacterium liquid subjected to inactivation treatment by formaldehyde in the step (2) is lower than that of the pasteurella multocida fermentation culture.

Further, alumina gel is adopted as an adjuvant in the step (3).

Further, in the step (3), the inactivated solution of the rabbit hemorrhagic disease virus baculovirus vector cell culture and the inactivated solution of pasteurella multocida are fully mixed and then added with an adjuvant.

Further, stirring at 10-30 r/min when the inactivated solution of the rabbit hemorrhagic disease virus baculovirus vector cell culture and the inactivated solution of the Pasteurella multocida are fully and uniformly mixed in the step (3).

Further, after the inactivated solution of the rabbit hemorrhagic disease virus baculovirus vector cell culture and the inactivated solution of the pasteurella multocida are fully and uniformly mixed in the step (3), an adjuvant is added and continuously and uniformly mixed.

Further, the inactivated solution of the rabbit hemorrhagic disease virus baculovirus vector cell culture and the inactivated solution of pasteurella multocida in the step (3) are fully and uniformly mixed, then an adjuvant is added, and stirring is carried out at 30-50 r/min.

Compared with the prior art, the invention has the advantages that the rabbit hemorrhagic disease virus antigen production adopts the full suspension cell culture technology, the production process does not use strong toxicity, the prepared antigen has high hemagglutination titer, good immunogenicity and advanced production process, and can be produced in a GMP workshop on a large scale; the pasteurella multocida antigen fermentation culture process is more mature, the immunogenicity of the prepared antigens among different batches is more stable, and the protection rate of the challenge efficacy test is further improved compared with the similar vaccines prepared by the prior art. The preparation process of the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida bivalent inactivated vaccine is simpler, the large-scale production and the product quality control are easy, the biological safety is good, the product quality is better, and the production cost is further reduced. The vaccine prepared by the method has better safety, higher immune challenge protection rate and better immune period and retention period than the similar vaccines prepared by the prior art.

Drawings

FIG. 1 is a flow chart of a preparation method of a rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease combined inactivated vaccine according to an embodiment of the invention.

Detailed Description

The present invention is not limited to the following embodiments, and the features and steps described in the present invention can be arbitrarily combined and can be performed in different orders without being limited to those described below.

Sources of virus seeds and strains: recombinant rabbit hemorrhagic disease virus VP60 baculovirus (BAC-VP60 strain), hereinafter referred to as recombinant baculovirus, has been deposited in China center for type culture Collection in 26.6.2018, with the address of Wuhan, Wuhan university, zip code 430072, with the preservation number of CCTCC V201833; the pasteurella multocida is C51-17 strain, which is preserved in China center for type culture Collection in 2018, 6 and 26 months, and the address is Wuhan, Wuhan university, postcode 430072, the preservation number is: CCTCC M2018401.

Example 1 recombinant Rabbit hemorrhagic disease Virus VP60 protein antigen cell culture tank suspension culture method

Culturing Sf9 cells subjected to suspension culture at 27-28 ℃ for 24-48 hours at 90-120 r/min, continuously carrying out passage amplification, wherein the passage ratio is 1: 2-1: 4, inoculating the cells into a cell culture tank, firstly reaching the minimum culture capacity of the culture tank, then continuously culturing and adding an insect cell culture medium, and controlling the parameters of the cell culture tank within a normal range in the process of culturing the cells by using the cell culture tank: the temperature is 27-28 ℃, the pH value is 6.0-6.2, the Dissolved Oxygen (DO) is 50-60%, and the stirring speed is 50-70 r/min. When the cells in the cell culture tank reach a certain density (about 2X 10)⁶One/ml) is added, the recombinant baculovirus is inoculated according to the proportion of 1 percent for virus maintenance culture, and the parameters of temperature, pH value, DO, stirring speed and the like are controlled in a proper range. And 4-5 days after inoculation, when the content of the pathological cells reaches more than 85%, and then cell culture can be obtained. Through the multiple culture experiments, the hemagglutination titer of 1% human O-type erythrocytes is tested on the culture product, the hemagglutination titer result of the culture product is shown in Table 1, and the hemagglutination titer (HA) of the cell culture product is not lower than 1: 1024.

TABLE 1 results of hemagglutination titer (HA) of the cell culture tank culture product of recombinant rabbit hemorrhagic disease virus VP60 baculovirus

Volume of culture Medium (liter)	Time to collect poison (day)	HA(log₂)
			5	5	10
5	5	11
			6	4	10
6	5	11
			40	4	10
40	5	11
			50	5	11

EXAMPLE 2 preparation of inactivated liquid of cell culture of Leptovirus baculovirus vector for Rabbit hemorrhagic disease

Adding a formaldehyde solution with the final concentration of 0.2% into the prepared recombinant rabbit hemorrhagic disease virus VP60 baculovirus cell culture, uniformly mixing, replacing a tank, inactivating at 37 ℃ for 18-24 hours, and stirring at the rotating speed of 50-70 r/min; or after replacing the bottle, inactivating the virus at 37 ℃ for 24 hours, shaking the virus once every 2 to 3 hours, and then obtaining the inactivated liquid of the rabbit hemorrhagic disease virus baculovirus vector cell culture, wherein the inactivated liquid is stored at 2 to 8 ℃ and is required to be not more than 6 months. During inactivation test, inoculating 1% of inactivated liquid of a baculovirus vector cell culture of the rabbit hemorrhagic disease virus to well-grown Sf9 cells, culturing at 27-28 ℃, continuously observing for 5 days without cell lesion, harvesting the cell culture, repeatedly freezing and thawing at the temperature of-20 ℃ for 3 times, performing blind first generation, inoculating well-grown Sf9 cells, and culturing for 5 days without cell lesion and hemagglutination of cell sap.

Example 3 fermentation culture method of Pasteurella multocida

Preparation of 3 pasteurella multocida fermentation broth

3.1 first-order seed propagation and identification, properly diluting pasteurella multocida C51-17 strain freeze-dried bacteria by using sterilized normal saline, streaking and inoculating the diluted bacteria to a Martin agar plate containing 4% of serum and 0.1% of lysed whole blood, culturing for 18-22 hours at 37 ℃, selecting at least 5 typical colonies, respectively inoculating a plurality of Martin agar inclined planes containing about 4% of serum and about 0.1% of lysed whole blood, and culturing for 18-22 hours at 37 ℃ to serve as first-order seeds. The product is stored at the temperature of 2-8 ℃ and the service life is not more than 7 days.

3.2 second-level seed propagation and identification, first-level seeds are taken and inoculated in Martin broth containing 0.5 percent of serum, the Martin broth is placed at 37 ℃ for 200r/min, shaking culture is carried out for 14-18 hours, samples are taken for pure inspection, the qualified Martin broth is used as second-level seeds, the second-level seeds are stored at 2-8 ℃, and the service life is not more than 4 days.

3.3 the culture of the bacterial liquid adopts a fermentation tank for aeration culture, 30 to 70 percent of fermentation medium and 0.01 to 0.02 percent of defoaming agent are filled according to the volume of the fermentation tank, after the culture medium is subjected to steam autoclaving, when the temperature is reduced to about 37 ℃, secondary seed liquid is inoculated according to 5 to 10 percent of the amount of the culture medium, simultaneously, serum is added according to 0.5 percent of the amount of the culture medium, the aeration quantity is stabilized at 15LPM, the stirring speed is 200 to 300r/min, the pH value is 7.2 to 7.6, the minimum Dissolved Oxygen (DO) is 0, and the aeration culture is carried out at 37 ℃ for about 10 hoursAnd (6) harvesting the culture solution. Performing pure inspection and viable bacteria count, wherein viable bacteria count per ml of culture broth should not be less than 1.2 × 10¹⁰And (4) CFU. The results of bacterial counts at different inoculum sizes and fermentation times are shown in Table 2, and the maximum viable count of about 10 hours can reach 1.2 × 10¹⁰CFU/ml above.

TABLE 2 results of counts of different inoculum doses of fermentative bacteria

EXAMPLE 4 preparation of Pasteurella multocida inactivated solution

Adding a formaldehyde solution according to 0.4 percent of the total amount of the pasteurella multocida solution, uniformly mixing, inactivating at 37 ℃ for 30-36 hours after tank replacement, and stirring at the rotating speed of 50-70 r/min to obtain the pasteurella multocida solution inactivated solution. And (4) storing the inactivation liquid at 2-8 ℃ for no more than 6 months.

Inactivation test: after inactivation, sampling, inoculating Martin agar containing about 4 percent of serum and about 0.1 percent of lysed whole blood and a Pasteurella multocida fermentation culture medium, culturing at 37 ℃ for 24 hours, and performing aseptic growth.

Example 5 bivalent vaccine antigen proportioning calculation and formulation

According to the hemagglutination titer of the actually produced cell culture (VP60 protein) and the bacteria content of the pasteurella bacteria liquid, the hemagglutination titer of the VP60 protein before inactivation in each ml of the vaccine is 1:256, and the bacteria number of the rabbit pasteurella multocida-containing C51-17 strain is 7.5 × 10⁹And calculating the distribution ratio of each group of the vaccine according to the CFU standard. And then mixing the qualified inactivated liquid of the cell culture of the rabbit hemorrhagic disease virus baculovirus vector and the inactivated liquid of the pasteurella multocida according to the proportion, stirring at a low speed (10-30 r/min) for 10-20 minutes, adding an aluminum hydroxide gel adjuvant according to the ratio of the antigen to the aluminum gel of 9:1, stirring at 30-50 r/min for 10-20 minutes, and fully and uniformly mixing to obtain the rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine. Quantitatively subpackaging, plugging, sealing and labeling. Table 3 shows the qualified rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid and the qualified rabbit hemorrhagic disease virus baculovirus vector cell cultureThe pasteurella multocida inactivated liquid is prepared into vaccines with different proportions, and the obtained 4 batches of rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida disease combined inactivated vaccines are obtained.

TABLE 3 vaccine components ratios and vaccine formulations (per ml)

Example 6 sterility test of Rabbit hemorrhagic disease baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine

The 4 batches of inactivated vaccine were randomly sampled and noted for representativeness, each batch was sampled by one percent of the number of bottles, but not less than 5 bottles, and not more than 10 bottles at most, and each bottle was separately tested. Shaking the vaccine to be detected uniformly without treatment, directly taking 1.0ml to inoculate 50ml thioglycollate medium (TG), culturing at 35-37 ℃, absorbing the culture after 3 days, inoculating 2 small tubes of TG, each small tube of 0.2ml, culturing 1 small tube at 35-37 ℃, culturing 1 small tube at 23-25 ℃, taking 0.2ml, inoculating 1 small tube of tryptone soy peptone liquid medium (TSB), culturing at 23-25 ℃ and culturing for 7 days. Samples from each spot-check were grown aseptically.

Example 7 safety test of Rabbit hemorrhagic disease baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine

The vaccines are fully shaken up, 5 healthy rabbits with the age of 35-42 days are taken as test groups, parts with loose neck skin of the rabbits are selected, injection parts are disinfected by 75% alcohol cotton balls, and each injection vaccine is 2.0 ml. Meanwhile, 5 rabbits in the same condition were used as controls without vaccination, and were continuously observed for 14 days to record mental states, diet, feces, local and systemic reactions, etc. of the two groups. The results show that all the test rabbits are healthy and alive, the mental state, diet, excrement and injection parts are well absorbed, no lumps exist, and the vaccine safety is good.

Example 8 efficacy test of Rabbit hemorrhagic disease baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine

8.1 fully and uniformly shaking the prepared rabbit hemorrhagic disease virus baculovirus vector and the combined inactivated vaccine of the pasteurella multocida disease, taking 12 healthy susceptible rabbits (the HI titer of rabbit hemorrhagic disease virus antibodies is not higher than 1:2, and the body weight is 1.75-2.0 kg) with 2-3 months of age, injecting 1.0ml of vaccine respectively, and simultaneously setting up 12 rabbits in a control group.

8.2 after 14 days of partial virus challenge immunization of rabbit hemorrhagic disease virus, 6 immunized rabbits are taken and are injected with the rabbit hemorrhagic disease virus Wanfan strain hepatovirus (the virus content is 10) subcutaneously at each neck together with 6 control rabbits⁴ LD₅₀Ml)1.0ml, and continuously observed for 7 days.

8.3 pasteurella multocida virulence determination A single typical colony of the recovered strain was picked and inoculated into Martin broth, cultured at 37 ℃ for 12-16 hours, and 1.0ml of the bacterial culture was serially diluted 10-fold with sterile physiological saline for bacterial counting. After 24 hours, the bacterial suspension was diluted with sterilized normal saline so that about 4, 6, 8, and 10 bacteria were contained in each ml of the diluted solution, and 6 test rabbits were subcutaneously injected into the neck at 1.0 ml/dose. And (4) counting bacteria of the bacteria liquid for attacking while inoculating the test rabbits, wherein the counting result is the actual number of the attacking bacteria. The observation was continued for 7 days and the number of test rabbit deaths was recorded.

8.4 preparation of challenge bacteria according to the toxicity test result, selecting a single typical colony of the recovered strain, inoculating the single typical colony to Martin broth, culturing at 37 ℃ for 12-16 hours, and taking 1.0ml of bacterial culture to serially dilute with 10 times of sterilized normal saline to count bacteria. After 24 hours, the bacterial suspension was diluted with sterilized physiological saline to adjust the Pasteurella multocida C51-17 strain to a Minimum Lethal Dose (MLD) according to the counting results. And (4) counting bacteria of the bacteria liquid for attacking the virus while inoculating the test rabbit, wherein the counting result is the actual number of the bacteria for attacking the virus.

8.5 after 21 days of partial virus challenge and immunization of the pasteurella multocida, 6 immunized rabbits are taken, and together with 6 control rabbits, 1.0ml (with the bacterial content of 5-10 CFU/ml) of pasteurella multocida C51-17 strain virulent bacterial liquid with the minimum lethal dose is injected subcutaneously into each neck part, and the continuous observation is carried out for 7 days.

Results 8.6 rabbit hemorrhagic disease virus fraction: the control rabbits all die, and the immune rabbits all keep alive; pasteurella multocida fraction: control rabbits all died, and immunized rabbits were at least 5 healthy.

Example 9 minimal immunization dose of Rabbit hemorrhagic disease Virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine

9.1 fully shaking the prepared rabbit hemorrhagic disease virus baculovirus vector and the prepared pasteurella multocida disease bigeminal inactivated vaccine uniformly, and subcutaneously injecting healthy susceptible rabbits with the age of 2-3 months into necks by 1.0ml, 0.5ml, 0.25ml and 0.125ml respectively (the HI titer of rabbit hemorrhagic disease virus antibody is not higher than 1:2, and the weight is 1.75-2.0 kg); at the same time, a control group rabbit was set.

9.2 after 14 days of immunization of rabbit hemorrhagic disease virus challenge test, 6 rabbits are respectively immunized by different doses of each batch of vaccine, 6 rabbits are controlled under the same condition, and the neck is respectively injected with rabbit hemorrhagic disease virus Wanumu strain hepatotoxin (the virus content is 10)⁴LD₅₀Ml)1.0ml, and continuously observed for 7 days. The results show that the 3 vaccine batches have the immunization dose of 0.25 ml/rabbit and more, the rabbit hemorrhagic disease virus is used for counteracting the virus, the immunized rabbits are all healthy, at least 4/6 healthy and alive, 0.125 ml/rabbit is at least healthy and alive, the control rabbits are all dead, the dead rabbits have the pathological changes typical of the rabbit hemorrhagic disease, and the human O-type erythrocyte agglutination tests of the dead rabbit liver suspension are all positive (Table 4).

TABLE 4 protective test results of different doses of bivalent inactivated vaccine rabbit hemorrhagic disease virus Wanumu strain liver toxicity attacking

9.3 after 21 days of immunization of the rabbit pasteurella multocida challenge test, 6 rabbits are respectively immunized by different dosages of each batch of vaccine, 6 rabbits are respectively injected with 1.0ml of pasteurella multocida C51-17 strain virulent strain with Minimum Lethal Dose (MLD) subcutaneously at the neck together with 6 control rabbits with the same condition, and the continuous observation is carried out for 7 days. The results show that 3 batches of vaccine have immune doses of 1.0ml and 0.5ml and are used for immunizing the immunized rabbits with rabbit pasteurella multocida, the immunized rabbits have at least 5/6 healthy lives, the immunized rabbits have at least 2/6 healthy lives in 0.25ml and 0.125ml, all control rabbits die, the dead rabbits have pathological changes typical of rabbit pasteurella multocida disease, and the live bacteria of the dead rabbits are separated and identified to be the rabbit pasteurella multocida (Table 5).

TABLE 5 different doses of bivalent inactivated vaccine rabbit pasteurella multocida C51-17 strain challenge protection test results

9.4 conclusion

The result of the minimum immune dose measurement of the rabbit hemorrhagic disease virus baculovirus vector and the pasteurella multocida disease bivalent inactivated vaccine shows that when the vaccine immunity is 0.25 ml/dose or more, the immune rabbit can be protected against the attack of the rabbit hemorrhagic disease virus virulent virus; when the vaccine is used for immunizing at a dose of 0.5 ml/rabbit and more, at least 5 immune rabbits are protected against the attack of the rabbit Pasteurella multocida C51-17 strain, which indicates that 0.5 ml/rabbit (the hemagglutination titer of the protein containing VP60 before inactivation is 1: 128, and the number of Pasteurella multocida is 3.75 multiplied by 10)⁹CFU) is the minimum immunization dose for the inactivated vaccine.

As the vaccine may have antigen loss (reduced titer) in the preservation period and ensures that the vaccine can effectively prevent rabbit hemorrhagic disease and rabbit pasteurella multocida disease in a longer immune period, in practical application, the immune dose of the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida disease combined inactivated vaccine (BAC-VP60 strain + C51-17 strain) is set as 1.0 ml/body (the hemagglutination titer of the VP60 protein before inactivation is 1:256, and the number of pasteurella multocida is 7.5 multiplied by 10⁹CFU)。

Example 10 immunization phase of Rabbit hemorrhagic disease baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine

10.1 immunization of vaccine 3 batches of rabbit hemorrhagic disease virus baculovirus vector, combined inactivated vaccine of pasteurellosis multocida (batch numbers: 201501, 201502 and 201503) and 1 batch of commercial combined inactivated vaccine of rabbit hemorrhagic disease and pasteurellosis multocida (batch number: 2015004) are respectively immunized for healthy rabbits with the age of 35-42 days, and injected subcutaneously at the neck part of the rabbit, wherein each dose is 1.0 ml; at the same time, a control group rabbit was set.

10.2 counteracting toxic pathogen and observing

10.2.1 Rabbit hemorrhagic disease virus challenge test 3, 4, 5, 6, 7 months after immunization, respectively taking 6 rabbits immunized with each batch of vaccine, and respectively injecting the Venus anhui strain liver virus (virus content 10) with rabbit hemorrhagic disease virus subcutaneously in neck together with 6 control rabbits under the same condition⁴LD50/ml)1.0ml, continuously observing for 7 days, recording the number of dead and protected animals, performing a dissection observation on dead rabbits and taking livers for a human O-type hemagglutination test. The results show that after 3 batches of vaccines are immunized, the immunized rabbits are all healthy and alive by using rabbit hemorrhagic disease viruses, the immunized rabbits of the same product die 1 rabbit 7 months after immunization, the control rabbits die completely (Table 6), the dead rabbits have pathological changes typical of the rabbit hemorrhagic disease viruses, and human O-type erythrocyte agglutination tests of dead rabbit liver suspensions are all positive.

TABLE 6 duration of vaccine immunization test results (Rabbit hemorrhagic disease virus challenge)

10.2.2 after the rabbit pasteurella multocida challenge test immunization, in 3, 4, 5, 6 and 7 months, 6 rabbits are respectively immunized with each batch of vaccine, 6 rabbits are controlled with the same conditions, 1.0ml of pasteurella multocida C51-17 strain virulent liquid with Minimum Lethal Dose (MLD) is subcutaneously injected into the neck of each rabbit respectively, 7 days are continuously observed, the number of dead and protected animals is recorded, and the dead rabbits are subjected to autopsy observation and bacterial separation and identification. The results show that when the pasteurella multocida C51-17 strain is used for virus challenge, at least 5/6 healthy rabbits are challenged at 3, 4, 5 and 6 months after immunization, at least 4/6 healthy rabbits are challenged at 7 months after immunization, the rabbits immunized by the similar product die in the immune period, 4 rabbits die after virus challenge at 7 months cannot produce enough protection, all 6/6 die control rabbits die, the dead rabbits have pathological changes typical of the pasteurella multocida disease, and the liver bacteria of the dead rabbits are separated and identified and are all the pasteurella multocida (Table 7).

TABLE 7 vaccine immunization duration test results (Rabbit pasteurella multocida challenge)

10.2.3 serum antibody titer determination of rabbit at 1, 2, 3, 4, 5, 6, and 7 months before and after immunization, collecting blood of control group rabbit, separating serum, and determining rabbit hemorrhagic disease virus HI antibody titer and rabbit Pasteurella multocida antibody OD₄₅₀The value is obtained. And (4) carrying out statistical analysis on the antibody detection results of all the sera of the rabbits from 1 to 7 months with the virus challenge in the 7 th month. The result shows that the average HI antibody titer of the rabbit hemorrhagic disease virus in the serum of the rabbit reaches 1: 21 month after the immunization^3.173 months after immunization, the antibody titer of rabbit hemorrhagic disease virus HI in rabbit serum reaches the peak, and then gradually decreases, and the average antibody level in the serum is still maintained at 1:2 in 7 months after vaccine injection³Above, the average HI titer of the rabbit serum antibody of the control group is 1:2⁰～1:2^0.33To (c) to (d); ELISA detection of OD (origin-destination) by rabbit Pasteurella multocida antibody in rabbit serum 1 month after immunization₄₅₀The value reaches 0.423, and the OD is detected by ELISA of rabbit pasteurella multocida antibody in rabbit serum 3 months after immunization₄₅₀The peak value reached, and then gradually decreased, and the mean antibody OD was measured by ELISA in the serum 6 months after the injection of the vaccine₄₅₀The value is still above 0.4, while the average ELISA detection OD of the rabbit serum antibody of the control group₄₅₀The values are between 0.140 and 0.159 (Table 8, Table 9).

In conclusion, the immune period of the baculovirus vector and the pasteurella multocida disease bigeminal inactivated vaccine for rabbit hemorrhagic disease can reach 7 months, and the immune period for rabbit pasteurella multocida disease can reach 6 months.

TABLE 8 results of antibody titers of rabbit hemorrhagic disease virus HI in vaccine immunization phase

TABLE 9 ELISA test results of rabbit Pasteurella multocida antibody in vaccine immunization phase

EXAMPLE 11 shelf-Life of the bivalent vaccine

The vaccines stored at 2-8 ℃ are respectively subjected to safety inspection and efficacy inspection after 3, 6, 9, 12, 15, 18 and 21 months. The results show that 3 batches of vaccines stored for different times are subjected to overdose safety tests on healthy rabbits of 35-42 days old, 1 batch of similar products stored for 12 months are subjected to overdose safety tests, and all rabbits are healthy and alive after continuous observation for 14 days. The result shows that the 3 batches of the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida disease bigeminal inactivated vaccine have good safety after being stored for 21 months, and the like products have good safety after being stored for 12 months. Respectively immunizing 12 healthy susceptible rabbits with 3 batches of vaccines and 1 batch of similar products which are stored for different times, respectively taking 6 immunized rabbits 14 days after immunization, and carrying out rabbit hemorrhagic disease virus challenge efficacy test on 6 control rabbits under the same conditions; at 21 days after immunization, 6 immunized rabbits were taken, and tested for pasteurella multocida challenge efficacy, along with 6 control rabbits under the same conditions. The results showed that after 3 vaccine immunizations, the immunized rabbits were challenged with rabbit hemorrhagic disease virus, and all the immunized rabbits were alive (table 10), and all the control rabbits died; the immunized rabbits were challenged with pasteurella multocida and the immunized rabbits were at least 5/6 healthy, while the control rabbit 6/6 died (table 11). The result shows that the rabbit hemorrhagic disease virus baculovirus vector and the pasteurella multocida disease bivalent inactivated vaccine are stored at the temperature of 2-8 ℃ and the storage life is at least 18 months, and the storage life of the commercially available rabbit hemorrhagic disease virus bivalent inactivated vaccine and the commercially available pasteurella multocida disease bivalent inactivated vaccine is 12 months at the temperature of 2-8 ℃.

TABLE 10 vaccine shelf life potency test results (Rabbit hemorrhagic disease virus challenge)

-: no test was performed.

TABLE 11 vaccine shelf life potency test results (Rabbit pasteurella multocida challenge)

-: no test was performed.

The results show that the shelf life of the rabbit hemorrhagic disease baculovirus vector and the pasteurella multocida disease bivalent inactivated vaccine exceeds the level of corresponding domestic similar products.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine are characterized in that antigen components in the bivalent inactivated vaccine are a full suspension culture of recombinant rabbit hemorrhagic disease virus VP60 baculovirus Sf9 cells and pasteurella multocida zymocyte liquid;

the rabbit hemorrhagic disease virus baculovirus vector is a recombinant rabbit hemorrhagic disease virus VP60 baculovirus BAC-VP60 strain, and the preservation number is CCTCCV 201833; the pasteurella multocida is C51-17 strain, and the preservation number is as follows: CCTCCM 2018401;

the preparation method of the rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine comprises the following steps:

(1) preparation of rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation liquid: inoculating recombinant rabbit hemorrhagic disease virus VP60 baculovirus into Sf9 cells for culturing, harvesting the recombinant rabbit hemorrhagic disease virus VP60 baculovirus cell culture when lesion cells reach more than 85%, inactivating the harvested cell suspension culture, wherein the rotating speed of the Sf9 cell culture is different from the culturing speed of the inoculated recombinant rabbit hemorrhagic disease virus VP60 baculovirus, and the temperature of the harvested cell culture for inactivation is higher than the temperature of the rabbit hemorrhagic disease virus VP60 baculovirus culture;

(2) preparation of pasteurella multocida inactivated solution: performing fermentation tank ventilation culture on the pasteurella multocida, and performing inactivation treatment on the fermentation broth by pure inspection of secondary seeds adopted by the pasteurella multocida fermentation broth during culture;

(3) preparing a rabbit hemorrhagic disease baculovirus vector and a pasteurella multocida disease bivalent inactivated vaccine: the antigen content is that per milliliter of vaccine contains VP60 protein before inactivation, the hemagglutination titer is not less than 1:256, and the number of pasteurella multocida is not less than 7.5 multiplied by 10⁹Preparing the vaccine according to the CFU standard, adding an adjuvant, and fully and uniformly mixing;

in the step (1), the culture medium used for culturing is an insect cell culture medium, the inoculation amount of the recombinant rabbit hemorrhagic disease virus VP60 baculovirus is 1% of the total amount of the culture medium, and the culture conditions are that the temperature is 27-28 ℃, the pH value is 6.0-6.2, the Dissolved Oxygen (DO) is 50% -60%, and the stirring speed is 50-70 r/min;

in the step (2), the pasteurella multocida is subjected to fermentation tank aeration culture, and the method specifically comprises the following steps: single colony of Pasteurella multocida was inoculated into horses containing serum and lysed whole bloodD, culturing an agar slant to obtain a first-level seed of the Pasteurella multocida; inoculating the primary seeds into a Martin broth containing serum, and culturing to obtain secondary pasteurella multocida seeds; filling 60-70% of fermentation medium and 0.01-0.02% of defoaming agent according to the volume of a fermentation tank, sterilizing the medium, inoculating secondary seed liquid according to 5-10% of the amount of the medium, adding serum, stirring at the rotating speed of 200-300 r/min and the pH value of 7.2-7.6, ventilating and culturing, and harvesting culture broth; the viable bacteria content of each ml of culture solution should not be less than 1.2 × 10¹⁰CFU。

2. The rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine as claimed in claim 1, wherein the adjuvant is aluminum hydroxide gel, and the ratio of the antigen to the aluminum hydroxide gel is 9: 1.

3. The rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine as claimed in claim 1, wherein after the rabbit hemorrhagic disease baculovirus vector cell culture obtained in the step (1) is obtained, hemagglutination titer determination is required, and the hemagglutination titer of the cell culture to 1% human O-type red blood cells is not less than 1: 512.

4. The rabbit hemorrhagic disease baculovirus vector, pasteurellosis multocida disease bivalent inactivated vaccine as claimed in claim 1, wherein in the step (1), the harvested cell culture is subjected to inactivation treatment by formaldehyde; and performing inactivation test on the inactivated rabbit hemorrhagic disease virus baculovirus vector cell culture inactivation solution: after the inactivation solution is inoculated to cells, no cytopathic effect exists, and hemagglutination does not exist in erythrocyte agglutination titer measurement.

5. The rabbit hemorrhagic disease baculovirus vector and pasteurella multocida disease bivalent inactivated vaccine as claimed in claim 1, wherein the harvested culture broth in step (2) is inactivated by formaldehyde, and subjected to inactivation test, and after inactivation, a sample is taken and inoculated with martin agar containing 4% of serum and 0.1% of lysed whole blood and a pasteurella multocida fermentation medium for culture, and the inactivated vaccine should be grown aseptically.