Method for efficiently and stably producing duck adenovirus type 3 vaccine antigen and application thereof
Through the LMH cell suspension culture method and inactivated emulsification formula, the problems of antigen production efficiency and purity of duck adenovirus type 3 vaccine in the existing technology are solved, and efficient, stable and low-cost vaccine production is achieved, suitable for large-scale production .
Patent Information
- Application Number
- CN202411183028.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-27
- Publication Date
- 2024-12-10
Smart Images
Figure BDA0005012755130000041 
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of veterinary biological products, and in particular to a method for efficiently and stably producing duck adenovirus type 3 vaccine antigen and an application thereof. Background Art
[0002] Duck adenovirus type 3 (DAdV-3) was first isolated from Muscovy ducks in Guangdong, my country in 2014. The main symptoms of ducks infected with DadV-3 are swelling and bleeding in the liver and spleen. The virus replicates at the highest titer in the liver, and the infected ducks can continue to excrete toxins into the environment. Not only can it spread rapidly horizontally through the oral-fecal route, but the mortality rate of DAdV-3 is as high as 40%, which makes its prevalence cause huge economic losses to my country's duck farming industry. Vaccine immunization is the most effective means to prevent adenovirus infection. The commonly used live or inactivated vaccines for waterfowl are mainly to infect duck embryos or primary cells derived from duck embryos with viruses, harvest the proliferated antigens, and then perform downstream purification and vaccine preparation. However, due to the variety, source, uniformity and individual differences of duck embryos, the quality of each batch of antigens has extremely high uncontrollable factors. In addition, the inoculation of duck embryos or the preparation of primary cells is cumbersome and cannot achieve efficient batching. It requires a lot of manpower and material resources and it is difficult to ensure high titer antigen output, which undoubtedly increases the cost of antigen preparation.
[0003] Therefore, in order to prepare high-titer, high-purity and cost-effective DAdV-3 vaccine antigens, establishing a stable and efficient antigen process suitable for DAdV-3 virus production is a prerequisite for ensuring the effectiveness of the vaccine. Summary of the invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for efficiently and stably producing duck adenovirus type 3 vaccine antigen and its application.
[0005] To achieve the above purpose, the technical solution designed by the present invention is as follows:
[0006] The present invention provides a method for efficiently and stably producing duck adenovirus type 3 vaccine antigen, comprising the following steps:
[0007] 1) After LMH cells are revived, they are inoculated into DMEM / F-12 liquid culture medium and cultured with shaking to obtain LMH cell suspension;
[0008] 2) inoculating the duck adenovirus type 3 seed virus into the LMH cell suspension obtained in step 1), and culturing by shaking to obtain a virus suspension;
[0009] 3) The virus suspension is then inoculated into a suspension reactor containing the LMH cell suspension obtained in step 1) to carry out expanded suspension culture to obtain duck adenovirus type 3 vaccine antigen.
[0010] Furthermore, in step 1), the density of LMH cells after LMH cell recovery is 1.5×10 6 cells / mL.
[0011] Furthermore, in step 1), the shaking culture conditions are:
[0012] The rotation speed was 120 r / min, the carbon dioxide concentration was 5%, the temperature was 37°C, and the culture time was 72 h.
[0013] Furthermore, in step 1), the cell density of the LMH cell suspension is 10 7 cells / mL.
[0014] Furthermore, in the step 2), DAdV-3 is inoculated and infected into LMH cell suspension at MOI=1; and the cell suspension is cultured with shaking for 48h-72h.
[0015] Furthermore, in step 2), the cell density of the virus suspension is 0.5×10 6 cells / mL-0.8×10 6 cells / mL, the titer of the virus suspension is 10 7.28 TCID 50 / mL.
[0016] Furthermore, in step 3), the virus suspension is inoculated into the LMH cell suspension at an MOI of 1; the suspension culture conditions are:
[0017] The pH value of the LMH cell suspension is 7.2±0.05, the temperature is 37.0°C; the dissolved oxygen is 38% to 50%, the stirring speed is 40r / min; and the culture time is 48h-72h.
[0018] Furthermore, in step 3), the live cell density of duck adenovirus type 3 vaccine antigen is 0.5×10 6 cells / mL-0.8×10 6 cells / mL, the titer of duck adenovirus type 3 vaccine antigen is 10 9 TCID 50 / mL.
[0019] The present invention also provides a method for preparing an inactivated vaccine of duck adenovirus type 3, which comprises adding the antigen obtained according to the above process into formaldehyde to obtain an antigen solution, inactivating the antigen, adding sterilized Tween 80 to prepare an aqueous phase of the vaccine, emulsifying the aqueous phase with white oil to prepare the vaccine.
[0020] Furthermore, in the antigen solution, the final volume of formaldehyde is 1.5‰; and the volume ratio of the aqueous phase to the white oil is 2:3.
[0021] Beneficial effects of the present invention:
[0022] (1) The method for producing duck adenovirus type 3 vaccine antigen provided by the present invention is simple to operate, has stable quality, can significantly improve the production efficiency of DAdV-3 antigen and save production costs;
[0023] (2) The method for producing duck adenovirus type 3 vaccine antigen provided by the present invention is to harvest the antigen from the cell supernatant of suspension culture, which has clear components and is free of serum and exogenous contamination. The titer of the harvested antigen can reach 10 9 TCID 50 / mL, direct dilution and vaccine preparation, further ensuring the purity and stability of the vaccine product;
[0024] (3) The method for producing duck adenovirus type 3 vaccine antigen provided by the present invention can be applied to the preparation of inactivated vaccines, has good safety, high immunogenicity, and is suitable for large-scale production of DAdV-3 vaccines in workshops. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 Results of vaccine injection site absorption 28 days after vaccination
[0026] Figure 2 Results of autopsy observation of ducks in the control group after infection
[0027] Figure 3 Results of autopsy observation of ducks in the vaccine-immunized group after challenge DETAILED DESCRIPTION
[0028] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.
[0029] Example 1 Screening of Condition Parameters for Suspension Culture of Chicken Hepatoma Cells (LMH Cells) (I) Effect of Density of Cell Suspension Culture on Cell Activity and Cell Proliferation
[0030] Chicken hepatoma cells (LMH cells) were cultured at 1.0×10 6 cells / mL、1.5×10 6 cells / mL and 3.0×10 6The cells were inoculated into a conical flask containing DMEM / F-12 liquid culture medium (purchased from Shanghai Yuchun Biotechnology Co., Ltd.) at three densities of 37°C and 120 r / min. 1 mL of samples were taken at 24 h, 48 h and 72 h, for a total of 9 samples, and the cell density and cell viability of each sample were measured.
[0031] The results showed that the density of cell suspension culture was a key factor affecting cell activity and cell proliferation. When the initial inoculation cell density was 1.5×10 6 cells / mL for 48 h, the harvested cell density was the highest, which could reach 4.0×10 6 cells / mL, and the cell viability was 98%; when the initial inoculation cell density was 1.5×10 6 cells / mL, and the cell density at harvest was the highest, reaching 1.0×10 7 cells / mL, and the cell viability was 98% (Table 1).
[0032] Table 1 Results of the optimal initial density test for LMH suspension cell passage
[0033]
[0034] (II) Effect of cell suspension culture density on cell density and cell viability
[0035] The optimal cell subculture initial density was 1.50×10 6 cells / mL. Under the conditions of a CO2 incubator at 37°C and a shaking speed of 120 r / min, 1 mL of samples were taken at 24 h, 48 h, 72 h, and 96 h to count the number of live cells and cell viability and determine the effect of culture time on cell growth.
[0036] The results showed that as the suspension culture time increased, the nutrients in the culture medium would be rapidly consumed, resulting in stagnation of cell growth and decreased cell viability; the cell density at 96 h was higher than that at 72 h, but the cell growth rate was slow and the cell viability decreased during 72 h-96 h, so the optimal culture time was 72 h (Table 2).
[0037] Table 2 Results of the optimal culture time test for LMH suspension cell subculture
[0038]
[0039] (III) According to the optimal initial cell density and culture time determined above, the cells were seeded at an initial density of 1.5×10 6cells / mL, and the culture temperature was 37℃. The shaking speeds of the shaker were set to 100r / min, 120r / min and 160r / min respectively. After 72h of culture, 1mL of sample from each group was taken to calculate the cell viability. Each group was repeated for 3 bottles and the average was taken.
[0040] The results showed that when the shaker speed was set to 120 r / min, the cell viability at 72 h was the highest (Table 3).
[0041] Table 3 Results of the optimal oscillation speed test for LMH suspension cells
[0042]
[0043] Example 2
[0044] A method for efficiently and stably producing duck adenovirus type 3 vaccine antigen comprises the following steps:
[0045] 1) Resuscitate LMH cells and then plate at a density of 1.5×10 6 LMH cells with a density of 1.0×10 cells / mL were inoculated in DMEM / F-12 liquid culture medium and cultured in suspension at a speed of 120 r / min, a carbon dioxide concentration of 5%, and a temperature of 37°C for 72 h to obtain a density of 1.0×10 7 cells / mL of LMH cell suspension;
[0046] 2) Duck adenovirus type 3 seed virus (DAdV-3 seed virus) was inoculated into the above LMH cell suspension at MOI = 1 and cultured with shaking for 48h-72h. When the cell density reached 0.5×10 6 cells / mL-0.8×10 6 cells / mL, the virus suspension was harvested and the titer was 10 7.28 TCID 50 / mL;
[0047] 3) The virus suspension was inoculated into a suspension reactor (75 L) containing the LMH cell suspension in step 1) at MOI = 0.5-1 for expanded suspension culture. During the culture process, the pH value of the LMH cell suspension was maintained at 7.2 ± 0.05, the temperature was 37.0 ° C, the dissolved oxygen was 38% to 50%, and the stirring speed was 40 r / min. When the viable cell density was 0.5 × 10 6 cells / mL-0.8×10 6 cells / mL, the vaccine antigen was harvested and its titer was 10 9 TCID 50 / mL.
[0048] Example 3
[0049] The method for preparing an inactivated vaccine of duck adenovirus type 3 comprises the following steps:
[0050] 1) The vaccine antigen obtained by the process of Example 2 was added to formaldehyde to obtain an antigen solution (the final volume of formaldehyde in the antigen solution was 1.5‰), inactivated at 37°C for 48h, and sterility and inactivation tests were performed according to the current Appendix III of the Chinese Veterinary Pharmacopoeia;
[0051] 2) The qualified antigens are first added to sterilized Tween 80 to prepare the water phase of the vaccine.
[0052] 3) Weigh the water phase and white oil in a volume ratio of 2:3 respectively, then add the white oil into the emulsification tank, start the shearing machine in the emulsification tank, and slowly add the prepared water phase. After emulsification for 30 minutes, aseptically package and obtain the finished vaccine.
[0053] 2. Analysis of vaccine absorption
[0054] One-week-old SPF ducklings were randomly divided into 2 groups:
[0055] a. A group of PBS immune (control group),
[0056] b. The above method is used to prepare the finished vaccine (immune group),
[0057] 0.5mL per feather, effective antigen content is 0.2×10 8 TCID 50 , observe the absorption of the vaccine 14 days after immunization.
[0058] The results showed that the finished vaccine was well absorbed, with only a small amount of unabsorbed residual spots observed ( Figure 1 ). 14 days after immunization, the virus was challenged and observed for 14 days. 8 / 10 of the control group developed disease ( Figure 2 ), manifested as enlarged and bleeding spleen, hemorrhage in the liver, and large areas of bleeding spots in the kidneys; the immune group was 10 / 10 protected ( Figure 3 ), spleen, liver and kidneys were normal without any lesions, and the immune protection was 100%.
[0059] From the above, it can be seen that a method for efficiently and stably producing duck adenovirus type 3 vaccine antigens is provided, which not only improves the content and purity of the antigen through suspension culture, thereby improving the effect of the vaccine, but also further reduces the cost of preparing inactivated vaccines of duck adenovirus type 3, and can be promoted as a production process for inactivated vaccines of duck adenovirus type 3.
[0060] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.
Claims
1. A method for efficiently and stably producing duck adenovirus type 3 vaccine antigen, characterized in that: The following steps are involved: 1) After LMH cells are revived, they are inoculated into DMEM / F-12 liquid culture medium and cultured with shaking to obtain LMH cell suspension; 2) inoculating the duck adenovirus type 3 seed virus into the LMH cell suspension obtained in step 1), and culturing by shaking to obtain a virus suspension; 3) The virus suspension is then inoculated into a suspension reactor containing the LMH cell suspension obtained in step 1) to carry out expanded suspension culture to obtain duck adenovirus type 3 vaccine antigen.
2. The method according to claim 1, characterized in that: In the step 1), the density of LMH cells after LMH cell recovery is 1.5×10 6 cells / mL.
3. The method according to claim 1, characterized in that: In the step 1), the shaking culture conditions are: The rotation speed was 120 r / min, the carbon dioxide concentration was 5%, the temperature was 37°C, and the culture time was 72 h.
4. The method according to claim 1, characterized in that: In step 1), the cell density of the LMH cell suspension is 10 7 cells / mL.
5. The method according to claim 1, characterized in that: In the step 2), DAdV-3 is inoculated and infected into LMH cell suspension at MOI=1; and the cell suspension is cultured with shaking for 48h-72h.
6. The method according to claim 1, characterized in that: In step 2), the cell density of the virus suspension is 0.5×10 6 cells / mL-0.8×10 6 cells / mL, the titer of the virus suspension is 10 7.28 TCID 50 / mL.
7. The method according to claim 1, characterized in that: In the step 3), the virus suspension is inoculated into the LMH cell suspension at an MOI of 1; the suspension culture conditions are: The pH value of the LMH cell suspension is 7.2±0.05, the temperature is 37.0°C; the dissolved oxygen is 38% to 50%, the stirring speed is 40r / min; and the culture time is 48h-72h.
8. The method according to claim 1, characterized in that: In the step 3), the live cell density of duck adenovirus type 3 vaccine antigen is 0.5×10 6 cells / mL-0.8×10 6 cells / mL, the titer of duck adenovirus type 3 vaccine antigen is 10 9 TCID 50 / mL.
9. A method for preparing an inactivated vaccine of duck adenovirus type 3, characterized in that: The method comprises adding the antigen obtained according to the process of claim 1 into formaldehyde to obtain an antigen solution, inactivating the antigen solution, adding sterilized Tween 80 to prepare the aqueous phase of the vaccine, emulsifying the aqueous phase with white oil to prepare the vaccine.
10. The preparation method according to claim 9, characterized in that: In the antigen solution, the final volume of formaldehyde is 1.5‰; the volume ratio of the water phase to the white oil is 2:3.