阅读说明：本技术 全悬浮鸭胚视网膜细胞在鸡产蛋下降综合症疫苗中的应用 (Application of full-suspension duck embryo retinal cells in vaccine for egg drop syndrome of chickens ) 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,具体而言,涉及全悬浮鸭胚视网膜细胞在鸡产蛋下降综合症疫苗中的应用。全悬浮鸭胚视网膜细胞在鸡产蛋下降综合症疫苗中的应用,将K-11株病毒接种到全悬浮鸭胚视网膜细胞中培养获得产蛋下降综合征病毒的抗原液,再将产蛋下降综合征病毒的抗原液与疫苗佐剂混合得到鸡产蛋下降综合症疫苗。将全悬浮鸭胚视网膜细胞应用于鸡产蛋下降综合症疫苗可以提高疫苗效价,且成本低。(The invention relates to the technical field of poultry vaccines, in particular to application of full-suspension duck embryo retinal cells in a vaccine for egg drop syndrome of chickens. The application of the full-suspension duck embryo retinal cells in the egg drop syndrome vaccine of chickens comprises the steps of inoculating K-11 strain virus into the full-suspension duck embryo retinal cells to culture to obtain antigen solution of egg drop syndrome virus, and mixing the antigen solution of egg drop syndrome virus with a vaccine adjuvant to obtain the egg drop syndrome vaccine of chickens. The application of the fully suspended duck embryo retinal cells in the chicken egg drop syndrome vaccine can improve the vaccine titer and has low cost.)

1. The application of the full-suspension duck embryo retinal cells in the vaccine for the egg drop syndrome of chickens is characterized in that: inoculating the K-11 strain virus into the fully suspended duck embryo retinal cells to culture to obtain antigen solution of the egg drop syndrome virus, and mixing the antigen solution of the egg drop syndrome virus with a vaccine adjuvant to obtain the egg drop syndrome vaccine.

2. Use according to claim 1, characterized in that: resuscitating and culturing the full-suspension duck embryo retinal cells, inoculating the K-11 strain virus into the cultured full-suspension duck embryo retinal cells, adding a DMEM (DMEM) culture medium for culturing, and then performing virus harvesting and freeze thawing.

3. Use according to claim 2, characterized in that: the recovery and culture of the fully suspended duck embryo retinal cells comprise the following steps: taking out the full suspension duck embryo retinal cells from a liquid nitrogen tank, rapidly melting the cells in a water bath at 37 ℃, centrifuging for 5 minutes at 200g, removing supernatant, suspending the full suspension duck embryo retinal cells by using 30ml of cell growth medium, and inoculating the full suspension duck embryo retinal cells to the cells125ml of the shake flask, placing the shake flask in a shaking table at a rotation speed of 160r/min and a temperature of 37 ℃ under 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.

4. The use of claim 3, wherein the whole suspension duck embryo retinal cells are expanded to 250ml shake flask, and then cultured until the cell density is 2.0 × 10⁶/-1.0×10⁷And/ml, inoculating the K-11 strain virus into the fully suspended duck embryo retinal cells.

5. Use according to claim 2, characterized in that: the inoculation amount of the K-11 strain virus is 0.0001-0.01 mol.

6. Use according to claim 2, characterized in that: the culture temperature of the K-11 strain virus after being inoculated into the fully suspended duck embryo retinal cells is 35-39 ℃.

7. Use according to claim 2, characterized in that: the culture time of the K-11 strain virus after being inoculated into the fully suspended duck embryo retinal cells is 3-5 days.

8. Use according to claim 2, characterized in that: the addition amount of the DMEM medium is 0-20% of the volume ratio of the culture solution.

9. Use according to claim 2, characterized in that: inoculating the K-11 strain virus into the cultured full-suspension duck embryo retinal cells, and putting the cells into a shaking table for culturing at the rotating speed of 160r/min and CO₂The concentration was 5%.

Technical Field

The invention relates to the technical field of poultry vaccines, in particular to application of full-suspension duck embryo retinal cells in a vaccine for egg drop syndrome of chickens.

Background

Egg Drop Syndrome (EDS) is a common infectious virus of laying hens caused by Egg Drop Syndrome Virus (EDSV), and although the lethality 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 production of vaccines related to egg drop syndrome in China mainly adopts the inoculation of duck embryos for culture and preparation of the vaccines, because SPF duck embryos are expensive and small in quantity, the actual production requirements cannot be met, but non-duck embryos cannot guarantee the source of the duck embryos, the duck embryos possibly contain other viruses, 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 were also reported to produce a vaccine against egg drop syndrome, but the virus titer was around 17log2, the immune antibody level after mating was between 10-12log2, and the HA titer was still low.

Disclosure of Invention

The invention aims to provide application of full-suspension duck embryo retinal cells in a vaccine for egg drop syndrome of chickens, and the full-suspension duck embryo retinal cells are applied to the vaccine for egg drop syndrome of chickens, so that the titer of the vaccine can be improved, and the cost is low.

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

The invention provides application of full-suspension duck embryo retinal cells in a vaccine for egg drop syndrome of chickens, which is characterized in that K-11 strain virus is inoculated into the full-suspension duck embryo retinal cells to be cultured to obtain antigen solution of the egg drop syndrome virus, and then the antigen solution of the egg drop syndrome virus is mixed with a vaccine adjuvant to obtain the vaccine for egg drop syndrome of chickens.

Further, in some embodiments of the invention, the fully suspended duck embryo retinal cells are firstly revived and cultured, then the K-11 strain virus is inoculated into the cultured fully suspended duck embryo retinal cells, and simultaneously DMEM medium is added for culture, and then the virus is collected and frozen and thawed.

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 the cells in a water bath at 37 ℃, centrifuging for 5 minutes at 200g, removing supernatant, suspending the fully suspended duck embryo retinal cells by using 30ml of cell growth solution, inoculating the fully suspended duck embryo retinal cells into 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 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 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 K-11 strain virus into the fully suspended duck embryo retinal cells.

Further, in some embodiments of the present invention, the virus of the above K-11 strain is inoculated in an amount of 0.0001 to 0.01 mol.

Further, in some embodiments of the invention, the above K-11 strain virus is cultured at a temperature of 35-39 ℃ after being inoculated into the fully suspended duck embryo retinal cells.

Further, in some embodiments of the invention, the above K-11 strain virus is cultured for 3-5 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 invention, the K-11 strain virus is inoculated into cultured whole suspension duck embryo retinal cells, and the cells are cultured in a shaking table 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:

thus, the K-11 strain virus is inoculated to the fully suspended duck embryo retinal cells to be cultured to obtain the antigen solution of the egg drop syndrome virus, the cost is low, and the obtained vaccine has high titer and strong immunocompetence.

The invention adopts full suspension cell detection to proliferate egg drop syndrome virus, the method is quick and simple, the cell source is clear and definite, and the invention avoids the immunogenicity influence caused by overhigh protein impurities caused by duck embryo production and also reduces the risk of exogenous virus caused by embryo source. The technology has short virus culturing time and high virus titer, and the vaccine produced by the technology has high antibody titer and 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 application of full-suspension duck embryo retinal cells in a vaccine for egg drop syndrome of chickens, which is characterized in that K-11 strain virus is inoculated into the full-suspension duck embryo retinal cells to be cultured to obtain antigen solution of the egg drop syndrome virus, and then the antigen solution of the egg drop syndrome virus is mixed with a vaccine adjuvant to obtain the vaccine for egg drop syndrome of chickens. The K-11 strain virus is inoculated to the fully suspended duck embryo retinal cells to be cultured to obtain the antigen solution of the egg drop syndrome virus, the cost is low, and the obtained vaccine has high titer and strong immunocompetence.

Further, in some embodiments of the invention, the fully suspended duck embryo retinal cells are firstly revived and cultured, then the K-11 strain virus is inoculated into the cultured fully suspended duck embryo retinal cells, and simultaneously DMEM medium is added for culture, and then the virus is collected and frozen and thawed. The operation is very simple, and the prepared vaccine has high titer and strong immunocompetence.

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 the cells in a water bath at 37 ℃, centrifuging for 5 minutes at 200g, removing supernatant, suspending the fully suspended duck embryo retinal cells by using 30ml of cell growth solution, inoculating the fully suspended duck embryo retinal cells into 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 250ml shake flasks, and the initial cell density is 1.0 × 10⁶And/ml, placing in a shaking table, and continuing culturing. Therefore, the full-suspension duck embryo retinal cells can be fully revived and cultured to form a full-suspension duck embryo retinal cell receptor culture medium with appropriate density, and the full-suspension duck embryo retinal cells are effectively utilized.

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 K-11 strain virus into the fully suspended duck embryo retinal cells. Such cell density is more favorable for cell proliferation.

Further, in some embodiments of the present invention, the virus of the above K-11 strain is inoculated in an amount of 0.0001 to 0.01 mol.

Further, in some embodiments of the invention, the above K-11 strain virus is cultured at a temperature of 35-39 ℃ after being inoculated into the fully suspended duck embryo retinal cells. Such an amount of inoculation allows the maximum utilization of the medium while allowing the medium to be loaded. Such a temperature is more favorable for the proliferation of cells.

Further, in some embodiments of the invention, the above K-11 strain virus is cultured for 3-5 days after being inoculated into the fully suspended duck embryo retinal cells. Such a cultivation time can avoid the shortage of the number of the proliferations due to the excessively short time.

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 cell proliferation is facilitated.

Further, in some embodiments of the invention, the K-11 strain virus is inoculated into cultured whole suspension duck embryo retinal cells, and the cells are cultured in a shaking table at a rotation speed of 160r/min and CO₂The concentration was 5%. Such culture conditions allow for more rapid cell propagation.

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

Test example 1: optimum inoculation cell density test for K-11 strain virus

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 respectively inoculating the viruses of the egg drop syndrome K-11 strains, simultaneously adding DMEM medium with the volume ratio of 15%, putting into a shaking table, and placing at 160r/min, 37 ℃ 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 test results of optimal cell density for inoculation of K-11 strains of virus

The results showed that the cell density was 8.0 × 10 when inoculated with the egg drop syndrome virus⁶At the concentration of the virus, the HA titer can reach 23log2, the virus content is the highest, namely 107.6TCID50/0.1ml, so the optimal inoculation density of the cells is 8.0 × 10⁶/ml。

Test example 2: optimum inoculation amount test of K-11 strain virus

3 250ml shake flasks were used to culture the fully suspended duck embryo retinal cells, and the cell density was 8.0 × 10⁶The virus strains of egg drop syndrome K-11 are inoculated with 0.01MOI, 0.001MOI and 0.0001MOI respectively, and DMEM medium with volume ratio of 15% is added into the virus strains, and the virus strains are placed into a shaking table at 160r/min, 37 ℃ 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 optimal inoculation amount test results for K-11 strains of virus

The result shows that the K-11 strain virus is inoculated at 0.001MOI, the HA titer can reach 22log2, the harvested virus content is slightly higher and is 107.8TCID50/0.1ml, so the optimal inoculation amount of the egg drop syndrome virus is determined as 0.001 MOI.

Test example 3: optimum inoculation temperature test for K-11 strain virus

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 every ml, inoculating the K-11 strain of egg drop syndrome according to 0.001MOI, simultaneously adding 15% DMEM culture medium by volume, placing into a shaking table, respectively culturing at 35 deg.C, 37 deg.C and 39 deg.C, with the rotation speed of the shaking table being 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 optimal incubation temperature test results for K-11 strains of disease

The results showed that the HA titer could be rapidly increased by culturing at 39 ℃ after inoculation of K-11, but the final virus titer was not as high as that of 37 ℃, so the optimal temperature for culturing K-11 was set at 37 ℃.

Test example 4: optimal virus receiving time of 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 × 10⁶The virus strain K-11 is inoculated according to 0.001MOI, DMEM medium with the volume ratio of 15% is added at the same time, the mixture is placed into a shaking table, the temperature is 35 ℃ and the concentration is 5% CO at 160r/min₂Culturing, 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 virus-harvesting time for K-11 strains of disease

The result shows that after the 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 107.6TCID50/0.1ml, so the optimal virus recovery time after the inoculation of the egg drop syndrome virus is determined as 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 reached⁶And/ml, inoculating the K-11 strain of egg drop syndrome according to 0.001MOI, simultaneously adding DMEM culture medium with the volume ratio of 0%, 10%, 15% and 20%, respectively, culturing for 96h at 37 ℃ under the condition of 5% CO2, collecting the toxin, freezing and thawing for 1 time, and determining the HA titer and the 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 when 15% of DMEM is added by volume, and are respectively 24log2 and 107.3TCID50/0.1 ml.

Test example 6: immunogenicity research of full-suspension duck embryo retinal cell virus and duck embryo virus

Taking the full-suspension duck embryo retina cell virus and the duck embryo virus, respectively adding formaldehyde solution with final concentration of 0.1%, inactivating for 48 hours at 37 ℃, respectively adding sterilized Tween-80 into the completely inactivated cell virus and the duck embryo virus, uniformly mixing to prepare water phase, and respectively preparing the cell virus oil emulsion inactivated vaccine and the duck embryo virus oil emulsion inactivated vaccine according to the proportion of 2: 1 of oil phase and 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 Total suspension Duck embryo retinal cytotoxicity and Duck embryo toxicity immunogenicity test results

The result shows that the HI antibody average value of the full-suspension duck embryo retina cytotoxic vaccine immunity group is higher than that of the duck embryo cytotoxic vaccine immunity group. Therefore, the full-suspension duck embryo retinal cell cytotoxicity has better immunogenicity,

the preparation method of the egg drop syndrome vaccine and the vaccine provided by the invention have the following beneficial effects: thus, the K-11 strain virus is inoculated to the fully suspended duck embryo retinal cells to be cultured to obtain the antigen solution of the egg drop syndrome virus, the cost is low, and the obtained vaccine has high titer and strong immunocompetence.

The invention adopts full suspension cell detection to proliferate egg drop syndrome virus, the method is quick and simple, the cell source is clear and definite, and the invention avoids the immunogenicity influence caused by overhigh protein impurities caused by duck embryo production and also reduces the risk of exogenous virus caused by embryo source. The technology has short virus culturing time and high virus titer, and the vaccine produced by the technology has high antibody titer and 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.