Adenovirus culture method and application thereof

Abstract

The invention provides a culture method and application of adenovirus, and relates to the technical field of biology. The culture method of the adenovirus provided by the invention comprises the following steps: inoculating chicken liver cancer cells into a serum-free SF501 culture medium according to the inoculation density of 0.50 * 10 < 6 >-0.80 * 10 < 6 > cells / mL, culturing for 72 hours, adding a fed-batch culture medium to adjust the cell density of the chicken liver cancer cells to 2.50 * 10 < 6 >-3.20 * 10 < 6 > cells / mL, inoculating the adenovirus according to the virus inoculation dosage of 0.05% (v / v)-0.5% (v / v) of the volume of the culture medium, and culturing the adenovirus. The culture method is simple and efficient, the production efficiency of the vaccine can be further improved, and the adenovirus can be better prevented and treated.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for culturing adenovirus and its application. Background Art

[0002] Avian adenoviruses belong to the genus Aviadenovirus of the family Adenoviridae and are non-enveloped double-stranded DNA viruses. Avian adenoviruses can be divided into three groups: Group I avian adenoviruses have a common group antigen and are divided into 5 types (FAV A-E) based on cross-neutralization tests, including 12 serotypes. The representative strain is serotype 4 Group I avian adenovirus; Group II FAV includes turkey hemorrhagic enteritis virus, pheasant marble spleen virus, and avian splenomegaly virus; Group III FAV only includes egg drop syndrome virus (EDSV-76). Group I avian adenoviruses are specifically divided into 5 genotypes, namely A, B, C, D, and E, and are divided into serotypes 1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10, and 11 according to serotypes; the corresponding relationship between genotypes and serotypes is A(1), B(5), C(4, 10), D(2, 3, 9, 11), E(6, 7, 8a, 8b). Avian adenoviruses are latent infections in the initial stage of infection, with a low mortality rate. In the later stage of infection, different diseases show different pathogenicities. Clinically, the diseases caused by avian adenoviruses mainly include gizzard erosion, inclusion body hepatitis, and hydropericardium-hepatitis syndrome, etc.

[0003] Vaccine is an effective method for preventing and controlling avian adenoviruses, and the production of vaccines involves the culture of avian adenoviruses. CN110237247A, CN111269892B, and CN113755454B respectively disclose a method for culturing adenovirus. However, these culturing methods all have the problems of long culturing time and low virus titer. Therefore, in order to accelerate the production and research and development of vaccines, it is necessary to find a more efficient method for culturing adenovirus.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a method for culturing adenovirus to solve the above technical problems.

[0006] The second object of the present invention is to provide the application of the above culturing method in the preparation of vaccines.

[0007] In order to achieve the above objects, the following technical solutions are specifically adopted:

[0008] In the first aspect, the present invention provides a method for culturing adenovirus, including the following steps:

[0009] According to 0.50×10 6 ~0.80×106 The chicken liver cancer cells were inoculated in serum-free SF501 medium at an inoculation density of cells / mL and cultured. After 72 h of culture, the feeding medium was added to adjust the cell density of the chicken liver cancer cells to 2.50×10 6 ~3.20×10 6 cells / mL. Then, adenovirus was inoculated at a virus inoculation dose of 0.05%(v / v)~0.5%(v / v) of the medium volume, and adenovirus culture was carried out.

[0010] As a further technical solution, both the chicken liver cancer cell culture and the adenovirus culture are carried out in a reactor;

[0011] Preferably, both the chicken liver cancer cell culture and the adenovirus culture are suspension cultures;

[0012] Preferably, the reactor is a 50L cell culture reactor.

[0013] As a further technical solution, the feeding medium is serum-free SF501 medium.

[0014] As a further technical solution, the pH of both the chicken liver cancer cell culture and the adenovirus culture is 6.8~7.2.

[0015] As a further technical solution, the temperature of the chicken liver cancer cell culture is 35-39℃;

[0016] Preferably, the temperature of the chicken liver cancer cell culture is 37℃.

[0017] As a further technical solution, the temperature of the adenovirus culture is 32-34℃;

[0018] Preferably, the temperature of the adenovirus culture is 33℃.

[0019] As a further technical solution, the time of the adenovirus culture is 48~51h.

[0020] As a further technical solution, the culture method includes:

[0021] (1) Cell culture: The suspension-type chicken liver cancer cells were inoculated in serum-free SF501 medium at an inoculation density of 0.50×10 6 ~0.80×10 6 cells / mL and cultured at 37℃ for 72 h;

[0022] (2) Adjusting cell density: According to the viable cell density at 72 h in the chicken liver cancer cell culture stage, fresh serum-free SF501 medium was supplemented to adjust the cell density to 2.50×10 6 ~3.20×10 6cells / mL;

[0023] (3) Virus inoculation: Inoculate adenovirus into the culture medium containing chicken liver cancer cells with the cell density adjusted in step (2) at a virus inoculation dose of 0.05% (v / v) to 0.5% (v / v). At this time, it is recorded as 0 h of virus culture;

[0024] (4) Virus culture: Culture the virus at 33 °C and then harvest the virus.

[0025] As a further technical solution, when harvesting the virus in step (4), the titer of the virus solution is 9.5 - 10.0 Log(TCID 50 / mL).

[0026] In the second aspect, the present invention provides the application of the above culture method in the preparation of vaccines.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The culture method of adenovirus provided by the present invention is simple and efficient. Only by culturing the virus for 48 - 51 h can a virus solution with a titer of more than 9.5 Log(TCID 50 / mL) be obtained. Applying the culture method provided by the present invention to the preparation of adenovirus vaccines can further improve the production efficiency of vaccines and better achieve the prevention and control of adenovirus. Specific embodiments

[0029] The following will describe the implementation plan of the present invention in detail in combination with the embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can be obtained as conventional products through commercial purchase.

[0030] In the first aspect, the present invention provides a culture method of adenovirus, including the following steps:

[0031] Inoculate chicken liver cancer cells into serum-free SF501 medium at an inoculation density of 0.50×10 6 ~0.80×10 6 cells / mL for culture. After 72 h of culture, add a feeding medium to adjust the cell density of chicken liver cancer cells to 2.50×10 6 ~3.20×10 6cells / mL, and then after inoculating adenovirus at an inoculation dose of 0.05% (v / v) - 0.5% (v / v) of the culture medium volume, adenovirus culture is carried out.

[0032] It should be noted that in the present invention, the inoculation density of chicken hepatoma cells can be, for example, but not limited to, 0.50×10 6 cells / mL, 0.60×10 6 cells / mL, 0.70×10 6 cells / mL or 0.80×10 6 cells / mL;

[0033] In the present invention, the cell density of chicken hepatoma cells can be adjusted to 2.50×10 6 cells / mL, 2.70×10 6 cells / mL, 2.90×10 6 cells / mL, 3.00×10 6 cells / mL or 3.20×10 6 cells / mL by adding a feeding medium;

[0034] In the present invention, the inoculation dose of adenovirus can be, for example, but not limited to, 0.05% (v / v) of the culture medium, 0.1% (v / v) of the culture medium or 0.5% (v / v) of the culture medium.

[0035] The adenovirus culture method provided by the present invention is simple and efficient, and a virus solution with a titer of more than 9.5 Log(TCID 50 / mL) can be obtained only through 48 - 51 h of virus culture. Applying the culture method provided by the present invention to the preparation of adenovirus vaccines can further improve the production efficiency of vaccines and better achieve the prevention and control of adenovirus.

[0036] In some alternative embodiments, the chicken hepatoma cells in the present invention are fully suspended cells.

[0037] In some alternative embodiments, both the chicken hepatoma cell culture and the adenovirus culture are carried out in a reactor;

[0038] Preferably, both the chicken hepatoma cell culture and the adenovirus culture are suspension cultures;

[0039] Preferably, the reactor is a 50 L cell culture reactor.

[0040] In some alternative embodiments, the volume of the chicken hepatoma cell culture is 16 - 20 L, and the rotation speed is 25 - 30 rpm;

[0041] The volume of virus culture is 45 - 50 L, and the rotation speed is 38 - 42 rpm.

[0042] In some alternative embodiments, the DO for the chicken hepatocarcinoma cell culture and adenovirus culture is 40% - 50%, and the aeration ratio is 0.01 - 0.1 VVM.

[0043] Through the research of the inventor, it is found that under these conditions, it is more suitable for adenovirus culture.

[0044] In some alternative embodiments, the feeding medium is serum - free SF501 medium.

[0045] In some alternative embodiments, the pH for the chicken hepatocarcinoma cell culture and adenovirus culture is 6.8 - 7.2.

[0046] By further optimizing and adjusting the pH of the chicken hepatocarcinoma cell culture and adenovirus culture, the titer of the virus solution obtained by culture is higher.

[0047] In some alternative embodiments, the temperature for the chicken hepatocarcinoma cell culture is 35 °C, 36 °C, 37 °C, 38 °C or 39 °C, preferably 37 °C;

[0048] In some alternative embodiments, the temperature for the adenovirus culture is 32 °C, 33 °C or 34 °C, preferably 33 °C.

[0049] By further optimizing and adjusting the temperature of the chicken hepatocarcinoma cell culture and adenovirus culture, the titer of the virus solution obtained by culture is higher.

[0050] In some alternative embodiments, the culture method includes:

[0051] (1) Cell culture: Inoculate suspension - type chicken hepatocarcinoma cells at an inoculation density of 0.50×10 6 - 0.80×10 6 cells / mL in serum - free SF501 medium, and culture the cells at 37 °C for 72 h;

[0052] (2) Adjust cell density: According to the viable cell density at 72 h in the chicken hepatocarcinoma cell culture stage, add fresh serum - free SF501 medium to adjust the cell density to 2.50×10 6 - 3.20×10 6 cells / mL;

[0053] (3) Virus inoculation: Inoculate adenovirus in the medium containing chicken hepatocarcinoma cells after adjusting the cell density in step (2) at a virus inoculation dose of 0.05% (v / v) - 0.5% (v / v). At this time, it is recorded as 0 h of virus culture;

[0054] (4) Virus culture: The virus is cultured at 33°C and then harvested.

[0055] In some alternative embodiments, the culture time of the adenovirus is 48 - 51 h.

[0056] The present invention can further refine the culture time by controlling the parameters of the adenovirus culture results. In some alternative embodiments, the specific method is as follows: Starting from 40 h of virus culture, samples are taken every 2 hours to detect the cell viability and virus titer respectively. When the cell density is below 2.00×10 6 cells / mL or the cell viability is lower than 60%, the virus is harvested.

[0057] In some alternative embodiments, when harvesting the virus in step (4), the titer of the virus solution is 9.5 - 10.0 Log(TCID 50 / mL).

[0058] Second, the present invention provides the application of the above culture method in the preparation of vaccines.

[0059] The culture method of the adenovirus provided by the present invention is simple and efficient. Applying it to the preparation of adenovirus vaccines can further improve the production efficiency of vaccines and better achieve the prevention and control of adenoviruses.

[0060] The following further illustrates the present invention through specific examples. However, it should be understood that these examples are only for more detailed illustration and should not be construed as limiting the present invention in any way.

[0061] It should be noted that the culture method of the present invention is applicable to all avian adenoviruses. The following experiments are conducted using avian adenovirus (Group I, Type 4) as an example.

[0062] Example 1

[0063] A culture method of an adenovirus is carried out in a 50 L reactor with the following culture steps:

[0064] (1) Cell culture: Suspended chicken liver cancer cells are inoculated at an inoculation density of 0.70×10 6 cells / mL in a serum-free SF501 medium (Bei'anji Biotechnology Co., Ltd.) and cultured at 37°C for 72 h, with cell counting every 24 h;

[0065] (2) Adjusting cell density: According to the viable cell density of LMH cells at 72 h of the culture stage, fresh serum-free SF501 medium is supplemented to adjust the cell density to 3.00×10 6 cells / mL;

[0066] (3) Virus inoculation: Adenovirus was inoculated into the above-mentioned medium containing LMH cells at an inoculation dose of 0.2% (v / v), and this was recorded as 0 h of virus culture;

[0067] (4) Virus culture: The virus was cultured at 33 °C. Starting from 40 h of virus culture, samples were taken every 2 hours to detect the cell viability and virus titer respectively.

[0068] (5) Virus harvest: The virus was harvested when the cell density was lower than 2.00×10 6 cells / mL, or when the cell viability was lower than 60%.

[0069] Among them, the culture volume of the seed cells: 16 - 20 L, 25 - 30 rpm; the virus culture volume 45 - 50 L, the rotation speed 38 - 42 rpm; the reactor maintained the DO value at 40% - 50%, the aeration ratio was 0.01 - 0.1 VVM, and the pH was 7.0.

[0070] Example 2

[0071] In step (1) of this example, the inoculation density of LMH cells was 0.50×10 6 cells / mL. In step (2), the cell density was adjusted to 2.50×10 6 cells / mL. In step (3), the inoculation dose was 0.05% (v / v). In step (4), the virus culture temperature was 32 °C, and the reactor pH was 6.8. The rest was the same as in Example 1.

[0072] Example 3

[0073] In step (1) of this example, the inoculation density of LMH cells was 0.80×10 6 cells / mL. In step (2), the cell density was adjusted to 3.20×10 6 cells / mL. In step (3), the inoculation dose was 0.5% (v / v). In step (4), the virus culture temperature was 34 °C, and the reactor pH was 7.2. The rest was the same as in Example 1.

[0074] Example 4

[0075] In step (2) of this example, fresh Medium A (Zhejiang Yishengke Biotechnology Co., Ltd., NF01d) was supplemented. The rest was the same as in Example 1.

[0076] Example 5

[0077] In step (2) of this example, fresh Medium B (Shanghai Duoning Biotechnology Co., Ltd., E1 additive) was supplemented. The rest was the same as in Example 1.

[0078] Example 6

[0079] In step (2) of this example, fresh C medium (Shanghai Duoning Biotechnology Co., Ltd., E4 additive) was supplemented, and the rest was the same as in Example 1.

[0080] Example 7

[0081] In step (2) of this example, fresh serum-free SF501 medium was not supplemented, and the rest was the same as in Example 1.

[0082] Example 8

[0083] In step (1) of this example, the seeding density of LMH cells was 0.40×10 6 cells / mL, and the rest was the same as in Example 1.

[0084] Example 9

[0085] In step (1) of this example, the seeding density of LMH cells was 0.90×10 6 cells / mL, and the rest was the same as in Example 1.

[0086] Example 10

[0087] In step (2) of this example, the cell density was adjusted to 2.00×10 6 cells / mL, and the rest was the same as in Example 1.

[0088] Example 11

[0089] In step (2) of this example, the cell density was adjusted to 3.50×10 6 cells / mL, and the rest was the same as in Example 1.

[0090] Example 12

[0091] In step (4) of this example, the virus culture temperature was 31°C, and the rest was the same as in Example 1.

[0092] Example 13

[0093] In step (4) of this example, the virus culture temperature was 35°C, and the rest was the same as in Example 1.

[0094] Example 14

[0095] In step (3) of this example, the virus inoculation dose was 0.01% (v / v), and the rest was the same as in Example 1.

[0096] Example 15

[0097] In step (3) of this example, the virus inoculation dose was 0.6% (v / v), and the rest was the same as in Example 1.

[0098] Example 16

[0099] In this example, the pH of the reactor is 6.5, and the rest is the same as in Example 1.

[0100] Example 17

[0101] In this example, the pH of the reactor is 7.5, and the rest is the same as in Example 1.

[0102] Example 18

[0103] In step (1) of this example, the seeding density of LMH cells is 0.60×10 6 cells / mL. In step (2), the cell density is adjusted to 2.50×10 6 cells / mL, and the pH of the reactor is 7.2. The rest is the same as in Example 1.

[0104] Example 19

[0105] In step (1) of this example, the seeding density of LMH cells is 0.50×10 6 cells / mL. In step (2), the cell density is adjusted to 3.20×10 6 cells / mL. In step (3), the virus inoculation dose is 0.5% (v / v). The rest is the same as in Example 1.

[0106] Example 20

[0107] In step (2) of this example, the cell density is adjusted to 3.20×10 6 cells / mL. In step (3), the virus inoculation dose is 0.05% (v / v), and the pH of the reactor is 7.2. The rest is the same as in Example 1.

[0108] Test Example 1

[0109] To demonstrate that the adenovirus culture method of the present application can effectively improve the virus titer of adenovirus strain culture, the virus titers of the virus liquids prepared in Examples 1 to 20 were detected and the culture time was observed. The specific results are shown in Table 1.

[0110] Table 1

[0111]

[0112]

[0113] As can be seen from Table 1, through the above culture method, the adenovirus described in the present invention has a suspension-type chicken hepatocarcinoma cell seeding density of 0.50×10 6 -0.80×10 6 cells / mL, and the virus-inoculated cell density is 2.50×10 6 ~3.20×10 6cells / mL, the virus inoculation dose is 0.05% (v / v) - 0.5% (v / v), the virus culture temperature is 32 - 34 °C, and the virus titer is as high as 9.5 Log(TCID 50 / mL) or above, and the harvesting time can be shortened to 48 h; among them, when the cell inoculation density is 0.50×10 6 -0.80×10 6 cells / mL, the virus-infected cell density is 2.50×10 6 ~3.20×10 6 cells / mL, the virus inoculation dose is 0.05% (v / v) - 0.5% (v / v), and the virus culture temperature is 33 °C, after culturing for 48 h, a virus solution with a virus titer of 9.8 Log(TCID 50 / mL) can be obtained. When cultured for 51 h, the virus titer can reach 10.0 Log(TCID 50 / mL). The culture method of the present application can greatly improve the culture efficiency of adenovirus, and the virus titer of the virus solution is higher.

[0114] Comparative Example

[0115] The culture method provided in Example 1 of the present invention was compared with Patent 1 (CN110237247A), Patent 2 (CN111269892A) and Patent 3 (CN113755454B), and the results are shown in Table 2.

[0116] Table 2

[0117]

[0118]

[0119] As can be seen from Table 2, the culture method provided by the present invention is simple and efficient, and a virus solution with a titer of 9.5 Log(TCID 50 / mL) or above can be obtained only by culturing the virus for 48 - 51 h, which is far superior to Comparative Patents 1 - 3.

[0120] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing an adenovirus, characterized in that, It includes the following steps: Inoculate chicken liver cancer cells in serum-free SF501 medium at an inoculation density of 0.50×10 6 ~0.80×10 6 cells / mL, and culture for 72 h. Then, add feeding medium to adjust the cell density of chicken liver cancer cells to 2.50×10 6 ~3.20×10 6 cells / mL. After that, inoculate adenovirus at an inoculation dose of 0.05% (v / v) - 0.5% (v / v) of the medium volume, and then carry out adenovirus culture.

2. The culturing method of the adenovirus according to claim 1, characterized in that The culture of chicken hepatoma cells and the culture of adenovirus are both carried out in a reactor; Preferably, the culture of chicken hepatoma cells and the culture of adenovirus are both suspension cultures; Preferably, the reactor is a 50L cell culture reactor.

3. The culturing method of the adenovirus according to claim 1, characterized in that, The feeding medium is serum-free SF501 medium.

4. The culturing method of the adenovirus according to claim 1, characterized in that, The pH of the culture of chicken hepatoma cells and the culture of adenovirus is 6.8 - 7.

2.

5. The culturing method of the adenovirus according to claim 1, characterized in that, The temperature of the culture of chicken hepatoma cells is 35 - 39°C; Preferably, the temperature of the culture of chicken hepatoma cells is 37°C.

6. The culturing method of the adenovirus according to claim 5, wherein, The temperature of the culture of adenovirus is 32 - 34°C; Preferably, the temperature of the culture of adenovirus is 33°C.

7. The culturing method of the adenovirus according to claim 1, characterized in that, The culture time of the adenovirus is 48 - 51h.

8. The culturing method of the adenovirus according to claim 1, characterized in that, The culture method includes: (1) Cell culture: Suspension-type chicken liver cancer cells were inoculated in serum-free SF501 medium at an inoculation density of 0.50×10 6 ~0.80×10 6 cells / mL and cultured at 37°C for 72 h; (2) Adjust the cell density: According to the viable cell density at 72 h in the chicken liver cancer cell culture stage, supplement fresh serum-free SF501 medium to adjust the cell density to 2.50×10 6 ~3.20×10 6 cells / mL; (3) Virus inoculation: Inoculate adenovirus into the medium containing chicken hepatoma cells with the cell density adjusted in step (2) at an inoculation dose of 0.05% (v / v) - 0.5% (v / v). At this time, it is recorded as 0h of virus culture; (4) Virus culture: Cultivate the virus at 33°C and then harvest the virus.

9. The culturing method of the adenovirus according to claim 8, wherein When harvesting the virus in step (4), the titer of the virus solution is 9.5 - 10.0 Log(TCID 50 / mL).

10. Use of the culture method according to any one of claims 1 - 9 in the preparation of a vaccine.

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