CN111150840B - Tetrad vaccine for poultry and preparation method and application thereof - Google Patents
Tetrad vaccine for poultry and preparation method and application thereof Download PDFInfo
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Classifications
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- A61K2039/70—Multivalent vaccine
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a fowl tetrad vaccine, and a preparation method and application thereof, wherein the fowl tetrad vaccine is a fowl newcastle disease, H9 subtype avian influenza, infectious bursal disease and fowl adenovirus tetrad vaccine, and the inactivated fowl newcastle disease virus La Sota strain, the inactivated H9 subtype avian influenza virus HB01 strain, the capsid protein VP2 of the infectious bursal disease virus and the structural protein Fiber2 of the fowl adenovirus are mixed to prepare an oil-water emulsion. The four-linked vaccine for poultry provided by the invention has the advantages of high safety, good stability, long immune protection duration, capability of providing long-acting maternal antibodies for offspring chicks after immunization of hens, extremely low endotoxin content, small immune side reaction, good compatibility of four antigens, no competition and expression inhibition among different antigens, and capability of achieving the same immune effect as that of respective monovalent inactivated vaccines by four components in the four-linked vaccine for poultry.
Description
Technical Field
The invention relates to the field of biological products for livestock, in particular to a tetrad vaccine for poultry and a preparation method and application thereof.
Background
In the chicken raising industry, avian influenza (H9 subtype) and newcastle disease are always in a higher incidence ratio, particularly, chickens and laying hens are in a weaker resistance stage, are extremely easy to be infected, and once the infection occurs, huge economic loss is caused to a breeding farm, and vaccine prevention is one of key measures for preventing and controlling the occurrence of the avian influenza (H9 subtype) and the newcastle disease in the breeding farm, and is also a commonly adopted mode in the current breeding farm.
The existing infectious bursal disease virus gene is continuously mutated, the infection cases of the super-strong strains are continuously appeared, the clinical cases of the immunological failure are more and more, the analysis reasons are mainly that the currently used attenuated vaccine strain is greatly different from the popular super-strong strains in gene and virulence level, the infection of the infectious bursal disease virus super-strong strains cannot be effectively prevented by using the conventional attenuated vaccine, in addition, the infection of the infectious bursal disease virus is urgent to be prevented and controlled in the current farm because the disease age is earlier and earlier, the moderate virulence vaccine is not used yet, and the infectious bursal disease virus is still infected.
The avian adenovirus (I group, type 4) has susceptibility to various chickens and ducks, the infection of the chickens is most common, the pericardial hydrops-hepatitis syndrome caused by the avian adenovirus is urgent in onset and quick in transmission, and the high death rate (more than 90%) of the chickens can be caused, so that the laying rate of the laying hens is reduced. At present, no specific therapy exists, and no commercial vaccine is available for prevention, so that the development of safe and effective vaccine capable of preventing the disease is extremely important.
The current commercial vaccines are mostly traditional vaccines, the selected strains are early in separation year, and have great difference from the current epidemic strains in gene and antigenicity (especially RNA viruses which are extremely easy to mutate), and due to the existence of immune deviation, even if a chicken farm is immunized repeatedly for a plurality of times, the development of epidemic situation is difficult to control, meanwhile, the susceptibility of stress and diseases is also aggravated, the production performance of chicken groups is reduced, the investment of manpower and material resources is increased, and the economic loss is further increased.
Disclosure of Invention
The aim of the scheme is to prepare four antigens of newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease (I group, type 4), and the four antigens are emulsified to prepare the four inactivated vaccines of newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease (I group, type 4), so that the prevention and control requirements of the poultry industry on newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease (I group, type 4) are met.
In a first aspect, the invention provides a four-way vaccine for poultry, which is prepared from a newcastle disease antigen portion, an H9 subtype avian influenza antigen portion, an infectious bursal disease antigen portion and an avian adenovirus disease antigen portion;
the H9 subtype avian influenza antigen part is an inactivated H9 subtype avian influenza virus HB01 strain.
The preservation number of the H9 subtype avian influenza virus HB01 strain is CCTCC NO: v202017, deposited on the chinese collection of university of armed forces at 01 and 07, with the deposit address: university of martial arts in chinese; the post code is: 430072, class designation: avian influenza virus (subtype H9) HB01 strain.
The H9 subtype avian influenza virus HB01 strain has agglutination (i.e. hemagglutination) on chicken erythrocytes, and the hemagglutination can be specifically inhibited by positive serum of avian influenza (H9 subtype), and the strain belongs to avian influenza H9N2 type through genotyping, and accords with the characteristics of the H9 subtype avian influenza virus.
Further, the avian adenovirus antigen part is derived from a recombinant escherichia coli Rosetta-rFiber2 strain expressing avian adenovirus Fiber2 protein, the newcastle disease virus antigen part is an inactivated newcastle disease La Sota strain, and the infectious bursal disease virus antigen part is derived from a recombinant escherichia coli Rosetta-rVP2 strain expressing infectious bursal disease virus VP2 protein.
Further, the fowl tetrad vaccine is composed of inactivated chicken newcastle disease virus La Sota strain, inactivated H9 subtype avian influenza virus HB01 strain, capsid protein VP2 of infectious bursal disease virus and structural protein Fiber2 of avian adenovirus.
The antigen component of the newcastle disease part selected by the invention is total-toxin inactivated antigen, and the strain is selected from classical strain La Sota strain of newcastle disease IV. The La Sota strain of the IV line of the newcastle disease is also a classical vaccine strain which is most commonly used for preventing and controlling the newcastle disease at present, and in recent years, the clinical isolation of the strain of the newcastle disease is generally less, and the prevention and control effect of the strain is fully demonstrated. The antigen component of the H9 subtype avian influenza part is also total-toxin inactivated antigen, and the strain is selected from H9 subtype avian influenza virus HB01 strain clinically separated in 2015, and has a close relationship with the H9 subtype avian influenza isolate popular in recent years (2014-2018), stable proliferation on chick embryo, high titer, good immunogen and potential for developing vaccine. The antigen component of the infectious bursal disease part is the capsid protein-VP 2 protein of the infectious bursal disease virus, and the capsid protein-VP 2 protein of the super-variant strain of the infectious bursal disease virus which is popular in recent years is used as the antigen of the infectious bursal disease part after soluble expression, purification and endotoxin removal by using a prokaryotic expression system. The antigen component of the avian adenovirus disease (group 1, type 4) part is Fiber2 protein, and the avian adenovirus (group 1, type 4) structural protein Fiber2 protein is used for carrying out soluble expression, purification and endotoxin removal by a prokaryotic expression system and then is used as the antigen of the avian adenovirus disease (group 1, type 4) part.
Further, the content of the inactivated Newcastle disease virus La Sota strain is 10 8.0 EID 50 /0.1ml～10 9.0 EID 50 0.1ml, wherein the content of the inactivated H9 subtype avian influenza virus HB01 strain is 10 6.0 EID 50 /0.1ml～10 8.0 EID 50 0.1ml of the capsid of the infectious bursal disease virusThe protein VP2 antigen has a agar-agar expansion titer of 1: 8-1: 32, wherein the content of structural protein Fiber2 of the avian adenovirus is 40-60 mug/0.1 ml.
Further, the content of the inactivated Newcastle disease virus La Sota strain is 10 8.0 EID 50 0.1ml, wherein the content of the inactivated H9 subtype avian influenza virus HB01 strain is 10 7.0 EID 50 0.1ml, capsid protein VP2 antigen of said infectious bursal disease virus having a agar spread titer of 1:16, the content of structural protein Fiber2 of the avian adenovirus is 50 mug/0.1 ml.
Further, the poultry tetrad vaccine is an oil-water emulsion.
The invention uses chicken newcastle disease virus La Sota strain and H9 subtype avian influenza virus A/chicken/Hubei/01/2015 (short for HB01 strain) to respectively inoculate susceptible chick embryo for culture, harvest chick embryo allantoic fluid, ultrafiltration concentration and formaldehyde inactivation to be used as antigen I and antigen II; the recombinant escherichia coli Rosetta (DE 3) -rVP2 strain expressing infectious bursal disease virus VP2 protein and the recombinant escherichia coli Rosetta (DE 3) -rFiber2 strain expressing avian adenovirus (group I, group 4) Fiber2 protein are respectively used as an antigen III and an antigen IV after fermentation culture, induced expression, thallus crushing, centrifugal removal of thallus fragments and purification; mixing the four antigens according to a certain proportion, and adding a mineral oil adjuvant for emulsification. Is used for preventing diseases caused by newcastle disease virus, H9 subtype avian influenza virus, infectious bursal disease virus and avian adenovirus (I group, 4 type).
In a second aspect, the present invention provides a method for preparing the four-way vaccine for poultry, comprising:
diluting inactivated chicken newcastle disease virus La Sota strain, inactivated H9 subtype avian influenza virus HB01 strain, capsid protein VP2 of infectious bursal disease virus and structural protein Fiber2 of avian adenovirus, and fully mixing to antigen liquid;
adding tween-80 to the antigen solution to form an aqueous phase and an oil phase in a ratio of 1:1 to 3, and then shearing and emulsifying. Preferably, the aqueous phase and the oil phase are combined in a ratio of 1:2, and mixing the components in proportion.
Further, the capsid protein VP2 of the infectious bursal disease virus and the structural protein Fiber2 of the avian adenovirus are both obtained by the soluble expression of the supernatant of the escherichia coli Rosetta strain through a constructed prokaryotic expression system.
Further, the oil phase is composed of the following components in parts by weight: 94 parts of white oil for injection, 1-2 parts of aluminum stearate and 6 parts of span 80.
The preferred preparation method of the components is as follows: 94 parts of white oil for injection (in milliliters) and 1 part of aluminum stearate (in grams) are added, stirred until the white oil is completely transparent, then 6 parts of span-80 parts (in milliliters) are added, and after mixing, the mixture is sterilized at 121 ℃ for 30 minutes and cooled to room temperature for standby.
The invention further provides application of the fowl tetrad vaccine and the preparation method thereof in simultaneously immunizing chickens with newcastle disease, avian influenza, infectious bursal disease and avian adenovirus.
The newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease four-way vaccine provided by the invention has the following beneficial effects:
1. the prepared vaccine H9 subtype avian influenza virus HB01 strain is named as A/chicken/Hubei/01/2015, is a strain separated in 2015, has higher homology with H9 subtype avian influenza strains (2014-2018 strains) popular in recent years, has stronger pertinence to the influenza strains, and can effectively prevent and control H9 subtype avian influenza by preparing the inactivated vaccine.
2. The VP2 protein of the infectious bursal disease part and the Fiber2 protein of the avian adenovirus disease (I group, type 4) part of the vaccine prepared by the invention realize the soluble expression of the supernatant in an escherichia coli expression system (wherein the VP2 protein of the infectious bursal disease part can also form virus-like particles), the structure and the function are similar to those of the natural state, and the immunogenicity of the vaccine is furthest reserved.
3. The VP2 protein and Fiber2 protein expressed by the invention have the advantages of high yield, high purity, good antigenicity, low endotoxin content, excellent antigen immune protection effect as a vaccine, and can effectively prevent infectious bursal disease (including infectious bursal disease virus super-strong variant) and avian adenovirus disease (I group, 4 type).
4. The vaccine prepared by the invention is an inactivated vaccine combining the traditional total-toxin inactivated antigen and the novel genetic engineering subunit antigen, has high safety, good stability and long immune protection duration, can provide long-acting maternal antibodies for offspring chicks after immunizing hens, has extremely low endotoxin content and small immune side reaction, has good compatibility of four antigens, and can achieve the same immune effect as the respective monovalent inactivated vaccine by four components in tetrad vaccine.
Drawings
FIG. 1 is a flow chart of the preparation of a newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease four-way vaccine provided in the embodiment 1 of the present invention;
FIG. 2 is an analysis chart of the HA gene nucleotide evolutionary tree of the H9 subtype avian influenza HB01 strain provided in example 3 of the present invention;
FIG. 3 is a diagram showing the homology analysis of the nucleotide sequences of the H9 subtype avian influenza HB01 strain and the HA gene of the existing H9N2 epidemic strain provided in example 3 of the present invention;
FIG. 4 is an analysis chart of the NA gene nucleotide evolutionary tree of the H9 subtype avian influenza HB01 strain provided in example 3 of the present invention;
FIG. 5 shows the nucleotide sequence homology analysis of the H9 subtype avian influenza HB01 strain provided in example 3 and the NA gene of the existing H9N2 epidemic strain.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a preparation method of a newcastle disease, avian influenza, infectious bursal disease and avian adenovirus disease four-way vaccine, and fig. 1 is a flow chart of the preparation method, and the specific steps include:
source of toxic (fungus) seeds for production
The recombinant escherichia coli Rosetta (DE 3) -rVP2 strain expressing the avian adenovirus (group I, type 4) Fiber2 protein and the recombinant escherichia coli Rosetta (DE 3) -rFiber2 strain expressing the avian adenovirus VP2 protein are all identified, stored and supplied by the pre-biological Co-Ltd of the Wuhan family.
Wherein, the preservation number of the H9 subtype avian influenza virus HB01 strain is CCTCC NO: v202017, deposited on the chinese collection of university of armed forces at 01 and 07, with the deposit address: university of martial arts in chinese; the postal code 430072 is named in the classification: avian influenza virus (subtype H9) HB01 strain.
The H9 subtype avian influenza virus HB01 strain has agglutination (i.e. hemagglutination) on chicken erythrocytes, and the hemagglutination can be specifically inhibited by positive serum of avian influenza (H9 subtype), and the strain belongs to avian influenza H9N2 type through genotyping, and accords with the characteristics of the H9 subtype avian influenza virus.
(II) preparation of production toxic (fungus) seed
1. Preparation of virus seed for producing newcastle disease virus La Sota strain: sterilizing physiological saline as 10 4 Multiple dilution, allantoic cavity inoculation of 10 day old SPF chick embryo, each embryo 0.1ml. Selecting chick embryos which die and have obvious lesions after 72-120 hours of inoculation, respectively harvesting chick embryo allantoic fluid, and filling the chick embryo allantoic fluid into a sterilization container. Mixing allantoic fluid with sterile test and 1% chicken erythrocyte agglutination value not lower than 1:512 (micro method), quantitatively packaging, and freeze preserving. The harvest date, the virus seed algebra and the like are noted.
2. Preparation of H9 subtype avian influenza virus HB01 strain production virus seed: the virus seeds are diluted 104 times by sterilized normal saline, and 10 day-old SPF chick embryos are inoculated in allantoic cavities, wherein each embryo is 0.1ml. And (5) respectively harvesting chick embryo allantoic fluid of the chick embryo 60-120 hours after inoculation, and filling the chick embryo allantoic fluid into a sterilization container. Will be tested sterile and will have a hemagglutination value of not less than 1 for 1% chicken erythrocytes: 256 Mixing (micro method) chick embryo liquid, quantitatively packaging, and freeze preserving. The harvest date, the virus seed algebra and the like are noted.
3. Preparation of a recombinant E.coli Rosetta (DE 3) -rVP2 strain producing strain expressing infectious bursal disease virus VP2 protein: inoculating freeze-dried Rosetta (DE 3) -rVP2 strain on LA culture medium (containing 10 mug/ml Kana) by streaking, culturing at 37 ℃ for 15 hours, selecting typical colony meeting the standard, inoculating the typical colony on LB culture medium (containing 10 mug/ml Kana), culturing at 37 ℃ by shaking table 200r/min until OD600nm is about 1.0, harvesting, and preserving bacterial liquid at 2-8 ℃ for no more than 14 days as first-stage seed bacteria; the first seed bacterial liquid of Rosetta (DE 3) -rVP2 strain is inoculated in LB culture medium (containing 10 mug/ml Kana) according to the volume ratio of 2%, and is cultured by a shaking table at 37 ℃ for 200r/min until OD600nm is about 1.0, and the second seed bacterial liquid is obtained after harvesting. The bacterial liquid is preserved at 2-8 ℃ for no more than 7 days.
4. Preparation of recombinant E.coli Rosetta (DE 3) -rFiber2 strain producing strain expressing avian adenovirus (group I, type 4) Fiber2 protein: inoculating freeze-dried Rosetta (DE 3) -rFiber2 strain on LA culture medium (containing 10 mug/ml Kana) by streaking, culturing at 37 ℃ for 15 hours, selecting typical colony meeting the standard, inoculating the typical colony on LB culture medium (containing 10 mug/ml Kana), culturing at 37 ℃ by shaking table 200r/min until OD600nm is about 1.0, harvesting, and taking the strain as first-stage seed strain, wherein the bacterial liquid is stored at 2-8 ℃ for no more than 14 days; the first seed solution of Rosetta (DE 3) -rFiber2 strain is inoculated in LB culture medium (containing 10 mug/ml Kana) according to the volume ratio of 2%, and is cultured by a shaking table at 37 ℃ for 200r/min until OD600nm is about 1.0, and the second seed solution is obtained after harvesting. The bacterial liquid is preserved at 2-8 ℃ for no more than 7 days.
(III) preparation of virus liquid for seedling preparation
Preparation of virus liquid for 1 chicken newcastle disease virus vaccine
1.1 inoculating production seed, and sterilizing with physiological saline as 10 4 Diluting by times, inoculating 9-11 days old susceptible chick embryo into allantoic cavity of each embryo, sealing pinhole, placing 36-37 deg.C for continuous incubation, and without turning egg.
1.2 incubating and observing 1 time per day of eggs after inoculation, discarding the dead chick embryo within 48 hours, taking out the dead chick embryo at any time after 48 hours, and storing at 2-8 ℃ until 120 hours. After 120 hours, the chick embryo is taken out completely no matter whether the chick embryo dies or not, the air chamber is up-standing, and the chick embryo is placed at the temperature of 2-8 ℃ for cooling for 4-24 hours.
1.3 harvesting, taking out cooled chick embryos, sterilizing egg shells, respectively harvesting chick embryo allantoic fluid, dividing a plurality of chick embryos into a group, placing the chick embryos into the same sterile container, respectively sampling to carry out a erythrocyte agglutination test, wherein the erythrocyte agglutination value of 1% of chicks is not lower than 1:512 And (trace method), and simultaneously carrying out aseptic inspection according to the annex of the current Chinese animal pharmacopoeia, wherein the aseptic growth is carried out, and the aseptic growth is preserved below-15 ℃ for standby.
1.4 concentration of virus liquidConcentrating chicken embryo allantoic fluid of chicken newcastle disease virus La Sota strain, removing large particle impurities in virus liquid, concentrating to 1/4 of original volume with ultrafiltration concentration system, sampling concentrated virus liquid, and measuring virus content (after 4 times dilution of concentrated virus liquid, virus content should be more than or equal to 10 per 0.1 ml) 8.0 EID 50 And (5) storing the concentrated virus solution at 2-8 ℃.
1.5 inactivation of virus liquid the allantoic fluid of the chick embryo which is qualified in detection is added into formaldehyde solution, so that the final concentration of the formaldehyde solution is 0.2%, and the mixture is stirred while being fully mixed, and is inactivated for 20 hours at 37 ℃, and then is preserved for standby at 2-8 ℃.
Preparation of virus liquid for 2H9 subtype avian influenza virus vaccine preparation
2.1 inoculating production virus seed, using sterilized normal saline to make 104-time dilution, inoculating 9-11 days old susceptible chick embryo into allantoic cavity of each embryo, every embryo is 0.1ml, sealing pinhole, placing at 36-37 deg.C, continuously incubating, and turning egg.
2.2 incubating and observing 1 time per day of eggs after inoculation, discarding the dead chick embryo within 24 hours, taking out the dead chick embryo at any time after 24 hours, and storing at 2-8 ℃ until 96 hours. After 96 hours, the chick embryo is taken out completely no matter whether the chick embryo dies or not, the air chamber is up-standing, and the chick embryo is placed at the temperature of 2-8 ℃ for cooling for 4-24 hours.
2.3 harvesting, taking out cooled chick embryo, sterilizing eggshells, respectively harvesting chick embryo allantoic fluid, dividing a plurality of chick embryos into a group, placing the groups in a same sterilization container, respectively sampling to perform a erythrocyte agglutination test, performing sterile test on 1% of chick erythrocyte agglutination value not less than 1:256 (micro method) according to the annex of the current Chinese veterinary pharmacopoeia, performing sterile growth, and preserving below-15 ℃ for later use.
2.4 concentration of virus liquid the chick embryo allantoic liquid of H9 subtype avian influenza HB01 strain is coarsely filtered, the large granule impurity in the virus liquid is removed, then the ultrafiltration concentration system is used for concentrating to 1/4 of original volume, after the concentrated virus liquid is sampled, the virus content is measured (after the concentrated virus liquid is 4 times diluted, every 0.1ml of virus content is more than or equal to 10) 7.0 EID 50 And (5) storing the concentrated virus solution at 2-8 ℃.
2.5 inactivating the virus liquid, adding the allantoic fluid of the chick embryo which is qualified in detection into formaldehyde solution, wherein the final concentration of the formaldehyde solution is 0.2%, stirring while adding the formaldehyde solution to fully mix the formaldehyde solution, inactivating the chick embryo for 24 hours at 37 ℃, and preserving the chick embryo at 2-8 ℃ for later use.
Preparation of 3 infectious bursal disease Virus VP2 protein antigen
3.1 culturing and inducing the bacterial liquid, inoculating the bacterial for production into a synthetic culture medium (containing 10 mug/ml Kana) according to the volume ratio of 2%, culturing at 36-37 ℃ in a fermentation tank, wherein the stirring speed is 200r/min, the Dissolved Oxygen (DO) is 10%, the pH is 7.2-7.3, after culturing under the condition until the OD600nm reaches about 60, adding isopropyl thiogalactoside (IPTG) to the final concentration of 0.1mmol/L, and after inducing and culturing at 16 ℃ for 14 hours, collecting the fermentation liquid.
3.2 after culturing the antigen-extracted bacterial liquid, the bacterial cells were collected by centrifugation, and after 3 times of resuspension and washing with PBS buffer (pH 7.4, 0.01M), the bacterial cell was crushed by pressure by resuspension with 10ml PBS buffer (pH 7.4, 0.01M) per gram of wet bacterial cell weight, and the supernatant was collected by centrifugation.
3.3 purification of protein antigen VP2 protein is purified by ion exchange method. The purified rVP2 protein is filtered and sterilized by a 0.22 mu m pore-size filter and then stored at 2-8 ℃.
Preparation of 4-avian adenovirus (group I, type 4) Fiber2 protein antigen
4.1 culturing and inducing the bacterial liquid, inoculating the secondary seeds into a synthetic culture medium (containing 10 mug/ml Kana) according to the volume ratio of 2%, culturing at 36-37 ℃ in a fermentation tank, wherein the stirring speed is 200r/min, the Dissolved Oxygen (DO) is 10%, the pH is 7.2-7.3, after culturing until the OD600nm reaches about 60 under the condition, adding isopropyl thiogalactoside (IPTG) to the final concentration of 1.0mmol/L, and after inducing and culturing at 36-37 ℃ for 6 hours, collecting the fermentation liquid.
4.2 after culturing the antigen-extracted bacterial liquid, the bacterial cells were collected by centrifugation, and after 3 times of resuspension and washing with PBS buffer (pH 7.4, 0.01M), the bacterial cell was crushed by pressure by resuspension with 10ml PBS buffer (pH 7.4, 0.01M) per gram of wet bacterial cell weight, and the supernatant was collected by centrifugation.
4.3 purification of protein antigen the protein of interest was purified by His tag affinity chromatography. The purified Fiber2 protein is filtered and sterilized by a 0.22 mu m pore filter and then stored at 2-8 ℃.
(IV) preparation of vaccine
1 preparation of oil phase 94 parts (in milliliters) of white oil for injection, 1 part (in grams) of aluminum stearate, stirring while adding until the white oil is completely transparent, adding 80-6 parts (in milliliters), mixing, sterilizing at 121 ℃ for 30min, and cooling to room temperature for standby.
2 water phase preparation, namely respectively diluting inactivated chicken newcastle disease virus, H9 subtype avian influenza virus, infectious bursal disease virus VP2 protein and avian adenovirus Fiber2 protein to the concentration required by vaccine preparation, and mixing according to the following steps of 1:1:1:1 volume ratio, and the content of the newcastle disease virus antigen in the antigen liquid after mixing is 10 8.0 EID 50 0.1ml of the antigen of the subtype H9 avian influenza virus with the content of 10 7.0 EID 50 In the case of a volume of/0.1 ml, infectious bursal disease virus VP2 protein antigen agar-agar titer is 1:16, the content of the avian adenovirus Fiber2 protein antigen is 50 mug/0.1 ml. Taking 96 parts of mixed antigen liquid, adding 4 parts of sterilized tween-80, and stirring for 20-30 min to completely dissolve the tween-80.
3 emulsification 2 parts of oil phase is injected into an emulsifying tank, 1 part of water phase is slowly added while stirring at a low speed, and shearing emulsification is carried out after the addition is completed.
And 4, subpackaging the emulsified vaccine quantitatively, and capping and sealing. Labeling and storing at 2-8 deg.c.
Example 2
In this example, the tetrad vaccine prepared in example 1 was tested, and the specific steps were as follows:
1 trait
1.1 appearance of milky homogeneous emulsion.
The 1.2 dosage form is water-in-oil type. Taking a cleaning straw, sucking a small amount of vaccine drops into cold water, wherein the first drop is in cloud-like diffusion, and all the drops should not be diffused later.
1.3 stability 10ml of vaccine is taken and added into a centrifuge tube, and the centrifugation is carried out for 15min at 3000r/min, and the water phase separated out from the bottom of the tube is not more than 0.5ml.
1.4 viscosity is measured according to the annex of the current Chinese animal pharmacopoeia, and the viscosity meets the regulations.
The 2-dose inspection is measured according to the annex of the current Chinese animal pharmacopoeia, and the specification is met.
3, the sterile test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the sterile growth is needed.
4 safety test 10 SPF chickens 3-4 weeks old, 0.6ml of each intramuscular or subcutaneous vaccine, and continuous observation for 14 days should not cause any local and systemic adverse reaction caused by the vaccine.
5 efficacy test
5.1 Newcastle disease portions 15 SPF chickens of 3-4 weeks of age, 10 of which were injected subcutaneously or intramuscularly with 12 μl (1/25 of the feathers) of vaccine, and 5 of which served as controls. Each chicken was injected with 0.2ml of Nakagaku (CVCC AV1611 strain) virus solution (virus content 10) into each muscle 21-28 days after inoculation 5.0 ELD 50 ). The control chickens should die entirely and the immunized chickens should be protected by at least 7 after 14 consecutive days of observation.
The 5.2H9 subtype avian influenza fraction was treated with 15 SPF chickens of 3 to 4 weeks of age, 10 of which were each injected subcutaneously or intramuscularly with 0.3ml of vaccine, and 5 of which served as controls. Each chicken wing is injected with 0.2ml of H9 subtype avian influenza virus HB01 strain virus liquid (virus content is 2X 10) by intravenous injection 21-28 days after inoculation 6.0 EID 50 ) Collecting throat swabs and cloaca swabs of each chicken on day 5 after virus attack, mixing the throat swabs and cloaca swabs of the same chicken into 1 sample, inoculating 5 SPF chick embryos of 10 days old to each sample through allantoic cavities, incubating and observing for 5 days, and determining the HA titer of chick embryo allantoic fluid by embryo, wherein the hemagglutination titer of the allantoic fluid of 1 chick embryo is not lower than 1:16 in 5 chick embryos inoculated to each sample, and judging that virus separation is positive; the chick embryo allantoic fluid with the blood coagulation value lower than 1:16 (micro method) is mixed and then is subjected to blind transmission once again for judgment. At least 9 viruses should be isolated as negative in immunized chickens and at least 4 viruses should be isolated as positive in control chickens.
5.3 infectious bursal disease portions 20 SPF chickens of 3-4 weeks of age, 10 of which were injected subcutaneously or intramuscularly with 0.3ml of vaccine, the other 10 serving as controls. 21-28 days after inoculation, all test chickens were orally infected with infectious bursal disease virus HN-14 strain (bursal tissue virus)) 0.2ml of virus solution (virus amount 2X 10) 4.0 ELD 50 ). After detoxification, the clinical manifestations of the test chickens are observed daily, and the morbidity is recorded. 72 hours after toxin attack, the bursa of Fabricius is dissected and killed, and the bursa disease is observed. At least 8 immunized chickens should be protected and have no bursal disease; the control chicken should have at least 8 diseases, and have obvious bursal disease (one or more than one diseases such as bursal enlargement or atrophy, hemorrhage, yellowing, jelly-like secretion, etc.).
5.4 avian adenosis (group I, type 4) in 20 chickens with 7-10 days old SPF, 10 of which were injected subcutaneously or intramuscularly with 0.3ml of vaccine, and 10 of which were used as controls. 21 days after inoculation, together with control chickens, each chicken leg was intramuscular injected with 0.2ml (2X 10 virus amount) of avian adenovirus (group I, 4) KQ-HB strain virus solution 5.0 TCID 50 ) For 10 consecutive days, at least 8 immunized chickens should be protected and at least 8 control chickens should die.
The measurement of the residual quantity of the formaldehyde is carried out according to the annex of the current Chinese animal pharmacopoeia, and the specification is met.
Through the vaccine efficacy test, the four-way vaccine of the embodiment 1 meets the standard of the test method, and the four antigen parts in the four-way vaccine of the embodiment 1 can exert the efficacy of the vaccine, and the conditions of competition among antigens, expression inhibition and the like are not generated. Meanwhile, adverse reactions aggravated by different solubilities, physical compatibility and stability among antigen components are avoided.
Example 3
1. Sequencing and sequence analysis of H9 subtype avian influenza virus HB01 strain
According to the genome sequence of the avian influenza virus of the GeneBank database, designing primers to amplify HA and NA gene segments of HB01 strain, wherein the sequences of the primers are as follows:
HA-F：5’-GGGAGCAAAAGCAGGGG-3’(SEQ ID NO.1)；
HA-R：5’-GGAGTAGAAACAAGGGTGTTTT-3’(SEQ ID NO.2)；
NA-F：5’-GGGAGCAAAAGCAGGAGT-3’(SEQ ID NO.3)；
NA-R：5’-GGAGTAGAAACAAGGAGTTTTTT-3’(SEQ ID NO.4)。
after the HA and NA gene segments are sequenced, genetic evolution information of the HA and NA genes is analyzed by MEGA6 software respectively.
Comparing the HA gene sequence of HB01 strain with the nucleotide sequence of a comparative representative 16H 9 subtype avian influenza virus reference strain registered on GenBank, it can be seen that HB01 strain HAs low homology with the early epidemic strain and can reach 96.7% -97.3% with the recent epidemic strain (2015-2018 strain), which shows that the HA gene is continuously mutated with the passage of time, and the results are shown in FIG. 2 and FIG. 3, wherein the No. 1 is the H9 subtype avian influenza virus HB01 strain, and the No. 2-17 is the comparative representative 16H 9 subtype avian influenza virus reference strain registered on GenBank.
Comparing the NA gene sequence of HB01 strain with the nucleotide sequence of the representative 14 strain H9 subtype avian influenza virus registered on GenBank, it can be seen that HB01 strain NA presents the same rule as the HA gene: the homology with the early epidemic strains is low, and the homology with the recent epidemic strains (2014-2016 strain) can reach 95.9-97.8%; the results of the continuous variation of NA gene with time are shown in FIG. 4 and FIG. 5, and the comparison of H9 subtype avian influenza virus HB01 strain No. 1 in FIG. 5 and representative 14H 9 subtype avian influenza virus reference strain registered on GenBank No. 2-15.
2 immune protective effect
20 SPF chickens of 3-4 weeks old are randomly divided into 2 groups, 10 chickens are in each group, one group is provided with 0.3 ml/feather of inactivated vaccine of H9 subtype avian influenza virus HB01 by subcutaneous immunization of neck, and the rest 1 group is not inoculated as a control group. The wing vein was collected 21 days after immunization, serum was separated, and the antibody level was measured. After blood sampling is completed 21 days after immunization, 5 chickens of the immune group and the control group are respectively subjected to intravenous injection of 0.2ml of H9 subtype avian influenza virus HB01 strain (the virus content is 2 multiplied by 10) 6.0 EID 50 ) On day 5 after challenge, each chicken swab was collected, virus isolation was performed by inoculating 10-day-old SPF chick embryos into the allantoic cavity, and the post-immunization challenge protection rate was calculated to obtain the results shown in Table 1. The immune group induces the organism to generate serum HI titer which is 10log2 against H9 subtype avian influenza virus HB01 strain 21 days after immunization, and uses H9 standard antigenThe average value of the HI titers of the serum is measured to be 4log2, and the protection rate of the H9 subtype avian influenza virus HB01 strain of the immune group after challenge reaches 100%.
From the above results, it is found that the H9 subtype avian influenza virus HB01 strain has good immunogenicity, but the antibody level measured by the H9 standard antigen is low, and the effect is poor, reflecting that the H9 subtype avian influenza virus HB01 strain has great difference from the H9 standard antigen strain antigenicity due to the variation of the strain.
TABLE 1 HI antibody titer determination and protection rate after immunization of HB01 strain
Experimental example 1
The four-way inactivated vaccine is respectively subjected to efficacy comparison research with 4 monovalent inactivated vaccines with the same antigen content of the corresponding parts.
The method comprises the following steps: randomly dividing 7-day-old SPF chicks into 3 groups, 10 chicks/group, immunizing four-way inactivated vaccine under the skin of the neck of the group 1, wherein each immunization is 0.3ml, the vaccine under the skin of the neck of the group 2 is a monovalent inactivated vaccine for the fowl adenovirus disease, each immunization is 0.3ml, and the group 3 is a control group; 90 SPF chickens of 3-4 weeks of age are randomly divided into 9 groups, the 1 st-6 th groups are immune groups, 10 th groups are each, the 7 th-9 th groups are control groups, and 10 th groups are each. The four-way inactivated vaccine is subcutaneously immunized on the neck of the 1 st and 3 rd groups, each of which is immunized with 0.3ml, the four-way inactivated vaccine is subcutaneously immunized on the neck of the 2 nd group, each of which is immunized with 12 mu l, and the monovalent inactivated vaccine for H9 subtype avian influenza is subcutaneously immunized on the neck of the 4 th group, each of which is immunized with 0.3ml; group 5 cervical subcutaneous immunization of newcastle disease monovalent inactivated vaccine 12 μl/dose, 6 cervical subcutaneous immunization of infectious bursal disease monovalent inactivated vaccine, 0.3ml each dose.
Results: the immune group of the quadruple inactivated vaccine is equivalent to the corresponding newcastle disease monovalent inactivated vaccine, the corresponding H9 subtype avian influenza monovalent inactivated vaccine, the corresponding infectious bursal disease virus VP2 protein monovalent inactivated vaccine and the corresponding avian adenovirus Fiber2 protein monovalent inactivated vaccine in terms of virus attack protection effect after immunization 21 days, and the virus attack protection rate is 100%. The results show that the four antigens in the newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenovirus disease four-way inactivated vaccine have good compatibility.
TABLE 2 toxicity counteracting protection Effect of Newcastle disease
TABLE 3 toxicity counteracting protective Effect of H9 subtype avian influenza
TABLE 4 infectious bursal disease virus counteracting protective Effect
TABLE 5 protective effects against avian adenovirus disease
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
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Claims (8)
1. The fowl tetrad vaccine is characterized by comprising an inactivated chicken newcastle disease virus La Sota strain, an inactivated H9 subtype avian influenza virus HB01 strain, a capsid protein VP2 of infectious bursal disease virus and a structural protein Fiber2 of avian adenovirus;
the preservation number of the H9 subtype avian influenza virus HB01 strain is CCTCC NO: v202017;
the avian adenovirus is group I type 4 avian adenovirus.
2. The avian tetrad vaccine according to claim 1, wherein the inactivated newcastle disease virus La Sota strain content is 10 8.0 EID 50 /0.1ml~10 9.0 EID 50 0.1ml, wherein the content of the inactivated H9 subtype avian influenza virus HB01 strain is 10 6.0 EID 50 /0.1ml~10 8.0 EID 50 0.1ml, capsid protein VP2 antigen of said infectious bursal disease virus having a agar spread titer of 1:8~1:32, wherein the content of structural protein Fiber2 of the avian adenovirus is 40-60 mug/0.1 ml.
3. The avian tetrad vaccine according to claim 2, wherein the inactivated newcastle disease virus La Sota strain content is 10 8.0 EID 50 0.1ml, wherein the content of the inactivated H9 subtype avian influenza virus HB01 strain is 10 7.0 EID 50 0.1ml, capsid protein VP2 antigen of said infectious bursal disease virus having a agar spread titer of 1:16, wherein the content of structural protein Fiber2 of the avian adenovirus is 50 mug/0.1 ml.
4. A four-way vaccine for poultry according to any one of claims 1-3, characterized in that the four-way vaccine for poultry is an oil-water emulsion.
5. A method of preparing a four-way vaccine for poultry according to any one of claims 1 to 4, comprising:
diluting inactivated chicken newcastle disease virus La Sota strain, inactivated H9 subtype avian influenza virus HB01 strain, capsid protein VP2 of infectious bursal disease virus and structural protein Fiber2 of avian adenovirus, and fully mixing to obtain antigen liquid;
adding tween-80 to the antigen solution to form an aqueous phase and an oil phase in a ratio of 1: and (3) mixing the materials according to the proportion of 1-3, and then shearing and emulsifying the materials.
6. The preparation method according to claim 5, wherein the capsid protein VP2 of the infectious bursal disease virus and the structural protein Fiber2 of the avian adenovirus are both obtained by supernatant soluble expression by a prokaryotic expression system constructed by E.coli Rosetta.
7. The preparation method according to claim 5 or 6, wherein the oil phase consists of the following components in parts by weight: 94 parts of white oil for injection, 1-2 parts of aluminum stearate and 6 parts of span 80.
8. Use of a tetrad vaccine for poultry according to any one of claims 1-4 or a method of preparation according to any one of claims 5-7 for the manufacture of a medicament for simultaneous immunization of chickens against newcastle disease, H9 subtype avian influenza, infectious bursal disease and avian adenoviruses.
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