阅读说明：本技术 一种鸡产蛋下降综合征疫苗的制备方法及疫苗 (Preparation method of egg drop syndrome vaccine and vaccine ) 是由 邢刚 丁莉 李成山 岳丰雄 左榕琳 肖倩 潘倩 于 2020-05-07 设计创作，主要内容包括：本发明涉及家禽疫苗的技术领域,具体涉及一种鸡产蛋下降综合征疫苗的制备方法及疫苗。鸡产蛋下降综合征疫苗,包括灭活的产蛋下降综合征病毒的抗原液和疫苗佐剂。此疫苗所用抗原液HA效价高,成本低。鸡产蛋下降综合征疫苗的制备方法,包括如下步骤：先将全悬浮鸭胚视网膜细胞复苏、培养,然后将鸡产蛋下降综合征病毒(K-11株)接种到培养好的全悬浮鸭胚视网膜细胞中,同时添加DMEM培养基进行培养,然后收毒、冻融。可以得到效价更高、成本更低的疫苗。(The invention relates to the technical field of poultry vaccines, in particular to a preparation method of a vaccine for egg drop syndrome and the vaccine. A vaccine for egg drop syndrome of chicken comprises an antigen solution of inactivated egg drop syndrome virus and a vaccine adjuvant. The antigen liquid used by the vaccine HAs high HA titer and low cost. The preparation method of the egg drop syndrome vaccine comprises the following steps: reviving and culturing the fully suspended duck embryo retinal cells, inoculating the egg drop syndrome virus (K-11 strain) to the cultured fully suspended duck embryo retinal cells, adding a DMEM culture medium for culturing, and then performing virus harvesting and freeze thawing. The vaccine with higher titer and lower cost can be obtained.)

1. A vaccine for egg drop syndrome, comprising: comprises inactivated antigen solution of egg drop syndrome virus and vaccine adjuvant.

2. The egg drop syndrome vaccine of claim 1, wherein: the antigen solution of the inactivated egg drop syndrome virus is obtained by inoculating the egg drop syndrome virus (K-11 strain) of chicken to a fully suspended duck embryo retinal cell for culture.

3. A method for preparing a vaccine for egg drop syndrome according to claim 1 or 2, characterized in that: the method comprises the following steps: reviving and culturing the fully suspended duck embryo retinal cells, inoculating the egg drop syndrome virus (K-11 strain) to the cultured fully suspended duck embryo retinal cells, adding a DMEM culture medium for culturing, and then performing virus harvesting and freeze thawing.

4. The production method according to claim 3, characterized in that: the recovery and culture of the fully suspended duck embryo retinal cells comprise the following steps: taking out the whole suspension duck embryo retina cells from the liquid nitrogen tank, rapidly melting in 37 deg.C water bath, centrifuging for 5 min at 200g, discarding supernatant, suspending the duck embryo retina cells with 30ml cell growth liquid, inoculating into 125ml shake flask, placing the shake flask in a shaking table at a rotation speed of 160r/min and a temperature of 37 deg.C and CO₂Culturing at 5% concentration for 2-3 days until the cell density reaches at least 6.0 × 10⁶When the cells are used for per ml, the fully suspended duck embryo retinal cells are expanded and passaged to 250ml shake flasks, and the initial cell density is 1.0 × 10⁶And/ml, placing in a shaking table, and continuing culturing.

5. The method according to claim 4, wherein the whole suspension of the duck embryo retinal cells is expanded to a 250ml shake flask, and then cultured until the cell density is 2.0 × 10⁶/-1.0×10⁷And/ml, inoculating the egg drop syndrome virus (K-11 strain) to the fully suspended duck embryo retinal cells.

6. The production method according to claim 3, characterized in that: the egg drop syndrome virus (K-11 strain) inoculation amount is 0.0001-0.01 MOI.

7. The production method according to claim 3, characterized in that: the culture temperature of the chicken egg drop syndrome virus (K-11 strain) after being inoculated into the fully suspended duck embryo retinal cells is 35-39 ℃.

8. The production method according to claim 3, characterized in that: the culture time of the egg drop syndrome virus (K-11 strain) after being inoculated into the fully suspended duck embryo retinal cells is 3-5 days.

9. The production method according to claim 3, characterized in that: the addition amount of the DMEM medium is 0-20% of the volume ratio of the culture solution.

10. The production method according to claim 3, characterized in that: inoculating the egg drop syndrome virus (K-11 strain) to the cultured fully suspended duck embryo retinal cells, and culturing in a shaking table at a rotation speed of 160r/min and CO₂The concentration was 5%.

Technical Field

The invention relates to the technical field of poultry vaccines, in particular to a preparation method of a vaccine for egg drop syndrome and the vaccine.

Background

Egg Drop Syndrome (EDS) is a common infectious disease of laying hens caused by Egg Drop Syndrome Virus (EDSV), and although the mortality rate of the disease is low, the egg yield of infected hens is seriously reduced, and the egg quality is influenced. The affected chickens mainly show symptoms of mass-production laying rate reduction, abnormal eggshells, egg body deformity, poor egg quality and the like. The disease can reduce the laying rate of laying hens by 30-50%, the breakage rate of eggs reaches 40%, the shell-free eggs and the soft-shell eggs can reach 15%, and serious economic loss is caused to the chicken industry.

At present, the vaccines related to the egg drop syndrome of chickens in China are mainly produced by inoculating duck embryos for culture and preparation, because SPF duck embryos are expensive and small in quantity, actual production requirements cannot be met, the non-immune duck embryos cannot guarantee the source of the duck embryos, other viruses are possibly contained in the duck embryos, the titer of the bred EDSV is greatly influenced, and the quality difference of the vaccines in different batches is huge. Meanwhile, the virus suspension cultured by the duck embryo contains a large amount of heterologous protein, so that the side effect of the vaccine is large.

In recent years, poultry vaccine related products are all strived to separate from embryo-derived production, and new production media, such as subunit vaccines and cell-derived vaccines, are searched. EB66 cells have also been reported to produce a vaccine against egg drop syndrome, but the viral titer is around 17log2, the immune antibody level after mating is between 10-12log2, and the antibody level is still relatively low.

Disclosure of Invention

The invention aims to provide a vaccine for egg drop syndrome, which HAs high antigen HA titer and low cost.

The invention also aims to provide a preparation method of the egg drop syndrome vaccine so as to obtain the vaccine with higher potency and lower cost.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The invention provides a vaccine for egg drop syndrome, which comprises an inactivated antigen solution of egg drop syndrome virus and a vaccine adjuvant.

Further, in some embodiments of the present invention, the antigen solution of the inactivated egg drop syndrome virus is obtained by inoculating the egg drop syndrome virus (K-11 strain) to the whole suspension duck embryo retinal cell culture.

The invention provides a preparation method of the egg drop syndrome vaccine, which comprises the following steps: reviving and culturing the fully suspended duck embryo retinal cells, inoculating the egg drop syndrome virus (K-11 strain) to the cultured fully suspended duck embryo retinal cells, adding a DMEM culture medium for culturing, and then performing virus harvesting and freeze thawing.

Further, in some embodiments of the present invention, the resuscitating and culturing of the fully suspended duck embryo retinal cells comprises the following steps: taking out the full suspension duck embryo retina cells from the liquid nitrogen tank, rapidly melting in 37 deg.C water bath, centrifuging for 5 min at 200g, removing supernatant, suspending the duck embryo retina cells with 30ml cell growth liquid, inoculating the full suspension duck embryo retina cells into 125ml shake flask, placing the shake flask in a shaking table at a rotation speed of 160r/min, a temperature of 37 deg.C and CO₂Culturing at 5% concentration for 2-3 days until the cell density reaches at least 6.0 × 10⁶When the cells are used for per ml, the fully suspended duck embryo retinal cells are expanded and passaged to 250ml shake flasks, and the initial cell density is 1.0 × 10⁶And/ml, placing in a shaking table, and continuing culturing.

Further, in some embodiments of the invention, after the above-mentioned fully suspended duck embryo retinal cells are expanded to 250ml shake flasks,continuing to culture until the cell density is 2.0 × 10⁶/-1.0×10⁷And/ml, inoculating the egg drop syndrome virus (K-11 strain) to the fully suspended duck embryo retinal cells.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated at 0.0001 to 0.01 MOI.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated into the fully suspended duck embryo retinal cells at a culture temperature of 35-39 ℃.

Further, in some embodiments of the present invention, the above-described egg drop syndrome virus (K-11 strain) is cultured for 3 to 5 days after being inoculated into the fully suspended duck embryo retinal cells.

Further, in some embodiments of the present invention, the DMEM medium is added in an amount of 0 to 20% by volume of the culture solution.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated into cultured whole suspension duck embryo retinal cells, and the whole suspension duck embryo retinal cells are subjected to shake culture at a rotation speed of 160r/min and CO₂The concentration was 5%.

The preparation method of the egg drop syndrome vaccine and the vaccine provided by the invention have the following beneficial effects:

the invention adopts the full-suspension cell to proliferate the egg drop syndrome virus, the method is quick and simple, the cell source is clear and definite, the immunogenicity is prevented from being influenced by overhigh protein impurities caused by using duck embryo production, and the risk of exogenous virus caused by the embryo source is also reduced. The technology has short virus culturing time and high virus titer, and the vaccine produced by the technology has high titer, higher economic benefit and market prospect.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.

The invention provides a vaccine for egg drop syndrome, which comprises an inactivated antigen solution of egg drop syndrome virus and a vaccine adjuvant. The prepared vaccine has higher titer, faster antibody growth speed after immunization and low cost.

Further, in some embodiments of the present invention, the antigen solution of the inactivated egg drop syndrome virus is obtained by inoculating the egg drop syndrome virus (K-11 strain) to the whole suspension duck embryo retinal cell culture. The antigen solution of the egg drop syndrome virus obtained by the method has low cost, and the obtained vaccine has high titer and good immune effect.

The invention provides a preparation method of the egg drop syndrome vaccine, which comprises the following steps: reviving and culturing the fully suspended duck embryo retinal cells, inoculating the egg drop syndrome virus (K-11 strain) to the cultured fully suspended duck embryo retinal cells, adding a DMEM culture medium for culturing, and then performing virus harvesting and freeze thawing. The preparation method is very simple, the cost is low by adopting the full-suspension duck embryo retinal cells, and the prepared vaccine has high titer and good immune effect.

Further, in some embodiments of the present invention, the resuscitating and culturing of the fully suspended duck embryo retinal cells comprises the following steps: taking out the fully suspended duck embryo retinal cells from a liquid nitrogen tank, rapidly melting in 37 ℃ water bath, centrifuging for 5 minutes at 200g, removing supernatant, suspending the fully suspended duck embryo retinal cells with 30ml of cell growth liquid, inoculating the fully suspended duck embryo retinal cells in a 125ml shake flask, placing the shake flask in a shaking table at the set rotation speed of 160r/min and the temperature of 37 ℃ and CO₂Culturing at 5% concentration for 2-3 days until the cell density reaches at least 6.0 × 10⁶When the cells are used for per ml, the fully suspended duck embryo retinal cells are expanded and passaged to 250mIn a shake flask, the initial cell density was 1.0 × 10⁶And/ml, placing in a shaking table, and continuing culturing. Therefore, the full-suspension duck embryo retinal cell can be fully revived and cultured to obtain the full-suspension duck embryo retinal cell receptor culture medium with proper density.

Further, in some embodiments of the present invention, the whole suspension of the duck embryo retinal cells is expanded to 250ml of shake flask, and then cultured until the cell density is 2.0 × 10⁶/-1.0×10⁷And/ml, inoculating the egg drop syndrome virus (K-11 strain) to the fully suspended duck embryo retinal cells. Such cell densities are more favorable for virus propagation.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated at 0.0001 to 0.01 MOI. Such an amount of inoculation allows the maximum utilization of the medium while allowing the medium to be loaded.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated into the fully suspended duck embryo retinal cells at a culture temperature of 35-39 ℃. Such temperatures are more favorable for virus propagation.

Further, in some embodiments of the present invention, the above-described egg drop syndrome virus (K-11 strain) is cultured for 3 to 5 days after being inoculated into the fully suspended duck embryo retinal cells. Such incubation times can peak the virus titer.

Further, in some embodiments of the present invention, the DMEM medium is added in an amount of 0 to 20% by volume of the culture solution. Thus, the nutrient in the culture medium is more sufficient, and the virus proliferation is facilitated.

Further, in some embodiments of the present invention, the egg drop syndrome virus (K-11 strain) is inoculated into cultured whole suspension duck embryo retinal cells, and the whole suspension duck embryo retinal cells are subjected to shake culture at a rotation speed of 160r/min and CO₂The concentration was 5%. Such culture conditions allow better cell growth and faster virus propagation.

The features and properties of the present invention are described in further detail below with reference to examples.

Test example 1: egg drop syndrome Virus (K-11 Strain) optimal inoculum cell Density test

Taking 4 250ml shake flasks to culture the fully suspended duck embryo retinal cells, and respectively culturing to the cell density of about 2.0 × 10⁶/ml、6.0×10⁶/ml、8.0×10⁶/ml、1.0×10⁷Then inoculating egg drop syndrome virus (K-11 strain), adding 15% DMEM culture medium, placing into shaker at 160r/min, 37 deg.C, and 5% CO₂And (5) culturing. Collecting toxin after 96h, freezing and thawing for 1 time, and measuring HA titer and virus content. The results are detailed in table 1.

TABLE 1 results of the optimal inoculation cell density test for the egg drop syndrome Virus (K-11 strain)

The results showed that the cell density was 8.0 × 10 when inoculated with the egg drop syndrome virus⁶At the time of/ml, the HA titer can reach 23log2, and the virus content is the highest and is 10^7.6TCID₅₀0.1ml, so the optimal seeding density of cells is 8.0 × 10⁶/ml。

Test example 2: optimum inoculation amount test of egg drop syndrome virus (K-11 strain)

3 250ml shake flasks were used to culture the fully suspended duck embryo retinal cells, and the cell density was 8.0 × 10⁶Perml, inoculating egg drop syndrome virus (K-11 strain) at 0.01MOI, 0.001MOI, and 0.0001MOI respectively, adding 15% DMEM medium, placing in shaker at 160r/min, 37 deg.C, and 5% CO₂And (5) culturing. Collecting toxin after 96h, freezing and thawing for 1 time, and measuring HA titer and virus content. The results are detailed in table 2.

TABLE 2 results of the experiment for the optimal inoculation amount of the egg drop syndrome virus (K-11 strain) of chickens

The results show that the egg drop syndrome of chickenThe virus (K-11 strain) is inoculated at 0.001MOI, the HA titer can reach 22log2, and the harvested virus content is slightly higher and is 10^7.8TCID₅₀0.1ml, so the optimal inoculum size for egg drop syndrome virus was set at 0.001 MOI.

Test example 3: optimum culture temperature test for egg drop syndrome virus (K-11 strain)

3 250ml shake flasks were used to culture the fully suspended duck embryo retinal cells, and the cell density was 8.0 × 10⁶When the virus is used for each ml, inoculating egg drop syndrome virus (K-11 strain) of chicken according to 0.001MOI, adding 15% DMEM culture medium, placing into shaking table, culturing at 35 deg.C, 37 deg.C, and 39 deg.C respectively, with shaking table rotation speed of 160r/min and CO₂The culture was carried out at a concentration of 5%. Samples were frozen and thawed 1 time at 48h, 72h, and 96h, respectively, and the HA titers were determined. The results are detailed in Table 3.

TABLE 3 results of the optimal culture temperature test for the egg drop syndrome Virus (K-11 strain)

As a result, the optimal temperature for culturing the K-11 strain was determined to be 37 ℃ because the HA titer could be rapidly increased by culturing at 39 ℃ after inoculation of the egg drop syndrome virus (K-11 strain), but the final virus titer was not as high as that of the culture at 37 ℃.

Test example 4: optimal virus-collecting time of egg drop syndrome virus (K-11 strain)

Taking 1 250ml of shake flask to culture the fully suspended duck embryo retina cells, and culturing the cells until the cell density reaches 8.0 × 10⁶Perml, inoculating egg drop syndrome virus (K-11 strain) at 0.001MOI, adding 15% DMEM medium, placing in shaker at 160r/min, 35 deg.C, and 5% CO₂Culturing, sampling 72h, 96h and 120h after inoculation, freezing and thawing for 1 time, and determining HA titer and virus content. The results are detailed in Table 4.

TABLE 4 test results of the optimal time to harvest egg drop syndrome Virus (K-11 strain)

The results show that after the egg drop syndrome virus (K-11 strain) is inoculated and cultured for 96h and 120h, the HA titer is not greatly different, and the virus content result shows that the culture time is slightly higher than 96h and can reach 10^7.6TCID₅₀0.1ml, so the optimal time to harvest the virus after inoculation of the egg drop syndrome virus is determined to be 96 h.

Test example 5: comparative test for DMEM addition

Taking 4 250ml shake flasks to culture the fully suspended duck embryo retinal cells, and culturing the cells to 8.0 × 10 when the cell density is reached⁶Perml, inoculating egg drop syndrome virus (K-11 strain) at 0.001MOI, adding DMEM medium at 0%, 10%, 15%, 20% by volume, and culturing at 37 deg.C with 5% CO₂Culturing for 96h under the condition, collecting toxin, freezing and thawing for 1 time, and measuring HA titer and virus content. The results are detailed in Table 5.

TABLE 5 comparative test results for DMEM addition

Tests of different addition amounts of DMEM show that the HA titer and the virus content of the harvested virus are the highest and respectively 24log2 and 10 when 15% of DMEM is added by volume^7.3TCID₅₀/0.1ml。

Test example 6: immunogenicity research of chicken egg drop syndrome cytotoxin and duck embryo virus

Taking the egg drop syndrome cytotoxin of chicken and the duck embryo virus, respectively adding a formaldehyde solution with the final concentration of 0.1%, inactivating for 48 hours at 37 ℃, respectively adding sterilized Tween-80 into the completely inactivated cytotoxin and the duck embryo virus, uniformly mixing to prepare a water phase, and respectively preparing the cytotoxin oil emulsion inactivated vaccine and the duck embryo virus oil emulsion inactivated vaccine according to the proportion of 2: 1 of an oil phase and the water phase.

20 SPF chickens of 35 days old are randomly and averagely divided into 2 groups and 10 groups, the cytotoxin oil emulsion inactivated vaccine and the duck embryo cytotoxin oil emulsion inactivated vaccine are respectively injected subcutaneously into the neck, 0.5ml of each group, and 10 chicken non-immunized are taken as a control. On 21, 28 and 35 days after immunization, test chicken blood is collected to separate serum, and the HI antibody in the serum is detected by EDSV-HI antigen. The results are shown in Table 6.

TABLE 6 immunogenicity test results for egg drop syndrome cytotoxic and duck embryo toxic

The result shows that the HI antibody average value of the cytotoxic vaccine immunity group is higher than that of the duck embryo virus vaccine immunity group. Therefore, the cytotoxic immunogenicity is better.

The preparation method of the egg drop syndrome vaccine and the vaccine provided by the invention have the following beneficial effects:

the invention adopts the full-suspension cell to proliferate the egg drop syndrome virus, the method is quick and simple, the cell source is clear and definite, the immunogenicity is prevented from being influenced by overhigh protein impurities caused by using duck embryo production, and the risk of exogenous virus caused by the embryo source is also reduced. The technology has short virus culturing time and high virus titer, and the vaccine produced by the technology has high titer, higher economic benefit and market prospect.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.