Culture method of porcine deltacoronavirus

Abstract

The invention provides a method for culturing porcine deltacoronavirus, and relates to the technical field of microorganism amplification culture. According to the culture method of the porcine deltacoronavirus, firstly, host cells are subjected to cell culture in a reactor to obtain a culture solution A, and then in the virus culture stage, an existing culture method of the porcine deltacoronavirus is improved through the steps of adding pancreatin twice, controlling the virus inoculation amount and the like. Through detection, the culture method provided by the invention can effectively improve the virus titer of porcine deltacoronavirus culture.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial expansion culture, and in particular to a culture method of porcine deltacoronavirus. Background Art

[0002] Porcine deltacoronavirus disease is an intestinal infectious disease caused by porcine deltacoronavirus (PDCoV) with watery diarrhea, vomiting, dehydration, and loss of appetite as clinical symptoms. PDCoV has caused serious economic losses to the pig industry. Vaccine immunization is an effective means of preventing and controlling PDCoV. In recent years, mRNA vaccines have developed rapidly. Using the mRNA technology platform, vaccines against epidemic strains can be quickly developed with high protective efficacy. Therefore, the development of mRNA vaccines for PDCoV will open up a new situation for vaccine prevention and control.

[0003] At present, ST cells (pig testicular cells) are often used to propagate porcine delta coronavirus in suspension culture to produce inactivated vaccines for preventing porcine delta coronavirus disease. However, the virus titer of PDCoV virus obtained by the existing suspension culture method is low and cannot meet the market demand.

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

[0005] The purpose of the present invention is to provide a method for culturing porcine deltacoronavirus. The method of the present application can obtain a better virus titer, providing a basis for the research and vaccine preparation of porcine deltacoronavirus.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The present invention provides a method for culturing porcine deltacoronavirus, the method comprising:

[0008] (A) Cell culture: culturing host cells to obtain culture medium A;

[0009] (B) Virus culture: After culturing medium A for 24 hours, trypsin was added for the first time. Then, the virus was inoculated and cultured for another 24 hours. Trypsin was added for the second time. The virus was cultured for 68-74 hours and the virus suspension was harvested.

[0010] At the time of harvest: virus titer ≥ 8.5-10.0 Log (TCID50 / mL), activity rate 70-80%.

[0011] During the porcine deltacoronavirus inoculation process, the inoculation volume of the porcine deltacoronavirus venom is 0.1-3% of the volume of the culture medium A.

[0012] Furthermore, the cell culture and virus culture are both carried out in a cell reactor;

[0013] Preferably, the reactor is a 50L cell culture reactor or a 300L cell culture reactor.

[0014] Furthermore, the culture medium in the cell culture process is serum-free SF601 culture medium;

[0015] And / or, the culture medium in the virus infection culture process is serum-free SF601 culture medium.

[0016] Furthermore, the host cell is a ST cell.

[0017] Furthermore, during the cell culture, the initial cell density of the host cells is: 0.5-1.2×10 6 cells / mL;

[0018] When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.5 × 10 6 cells / mL.

[0019] Furthermore, the amount of pancreatic enzyme added for the first time in step (B) is 5 mg / L;

[0020] And / or, the amount of pancreatic enzyme added for the second time in step (B) is 5 mg / L.

[0021] Furthermore, the virus culture process in step (B) further includes: adding calcium ions after adding pancreatic enzyme for the first time and before inoculating the virus;

[0022] Furthermore, the calcium ions are added in the form of CaCl2·2H2O;

[0023] Preferably, after adding calcium ions, the Ca in the culture medium 2+ The concentration is 0.05-0.5 mmol / L. Furthermore, the parameters of the cell culture in step (A) include: a culture temperature of 37°C, a glucose concentration of 3-4 g / L in the culture medium, 40% dissolved oxygen, and a pH of 6.8-7.2;

[0024] And / or, the parameters of the virus culture in step (B) include: culture temperature of 35-37° C., glucose concentration in the culture medium of 3-4 g / L, dissolved oxygen of 40%, and pH of 6.8-7.2.

[0025] And / or, during the cell culture and virus culture process, the rotation speed of the reactor is 27-40 rpm.

[0026] Furthermore, the virus titer in the virus suspension in step (B) is 8.5 to 10.0 Log (TCID50 / mL).

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

[0028] The present invention provides a method for culturing porcine delta coronavirus, wherein the method first performs cell culture on host cells in a reactor to obtain culture solution A, and then adjusts the steps of adding trypsin twice and controlling the amount of virus inoculation in the virus culture stage, and it is detected that the virus titer of the existing porcine delta coronavirus culture can be effectively improved. At the same time, the method for culturing porcine delta coronavirus of the present application does not require liquid replacement culture in the cell culture and virus culture stages, which effectively simplifies the process flow of porcine delta coronavirus culture. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] According to one aspect of the present invention, a method for culturing porcine deltacoronavirus comprises:

[0031] (A) Cell culture: culturing host cells to obtain culture medium A;

[0032] (B) Virus culture: After culturing medium A for 24 hours, trypsin was added for the first time. Then, the virus was inoculated and cultured for another 24 hours. Trypsin was added for the second time. The virus was cultured for 68-74 hours and the virus suspension was harvested.

[0033] The harvesting standard is: virus titer ≥ 8.5-10.0 Log (TCID50 / mL); harvesting when the viability is 70-80%.

[0034] Preferably, the strain of porcine deltacoronavirus is of genotype GIIa;

[0035] During the inoculation process, the inoculation volume of porcine deltacoronavirus venom is 0.1-3% of the volume of culture medium A.

[0036] The present invention provides a method for culturing porcine deltacoronavirus. The method firstly cultures host cells in a reactor to obtain a culture solution A, and then effectively improves the virus titer of the existing porcine deltacoronavirus culture by adding trypsin twice and controlling the virus inoculation amount during the virus culture stage.

[0037] It should be noted that the addition of pancreatic enzymes during the virus culture stage in the present application can effectively enhance the proliferation ability and growth efficiency of the virus. Furthermore, the present application has obtained through experiments that the effect of adding pancreatic enzymes twice is optimal.

[0038] In a preferred embodiment of the present invention, the cell culture and virus culture are both carried out in a cell reactor;

[0039] Preferably, the reactor is a 50L cell culture reactor or a 300L cell culture reactor.

[0040] In a preferred embodiment of the present invention, the culture medium in the cell culture process is serum-free SF601 culture medium;

[0041] And / or, the culture medium in the virus infection culture process is serum-free SF601 culture medium.

[0042] In a preferred embodiment of the present invention, the host cell is a ST cell.

[0043] In a preferred embodiment of the present invention, the initial cell density of the host cells during cell culture is 0.5-1.2×10 6 cells / mL;

[0044] When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.5 × 10 6 cells / mL.

[0045] In a preferred embodiment of the present invention, the amount of pancreatic enzyme added for the first time in step (B) is 5 mg / L, and the amount of pancreatic enzyme added for the second time in step (B) is 5 mg / L.

[0046] As a preferred embodiment, the amount of pancreatin added can effectively improve the virus growth efficiency. However, if the concentration is higher than 5 mg / L, the high concentration of pancreatin will have a toxic effect on cells, leading to cell death. On the contrary, if the concentration is too low, the culture titer cannot be improved well.

[0047] In a preferred embodiment of the present invention, the virus culture process in step (B) further comprises: adding calcium ions after the first addition of pancreatic enzymes and before inoculation;

[0048] The calcium ions are added in the form of CaCl2·2H2O; after the calcium ions are added, the Ca 2+ The concentration is 0~0.5mmol / L.

[0049] The Ca content in the above culture medium 2+Typical but non-limiting preferred embodiments of the concentration and temperature are: 0.05mmol / L, 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L and 0.5mmol / L.

[0050] As a preferred embodiment, the addition of calcium ions can promote the increase of virus titer.

[0051] In a preferred embodiment of the present invention, the parameters of the cell culture in step (A) include: a culture temperature of 37° C., a glucose concentration of 3 to 4 g / L in the culture medium, 40% dissolved oxygen, and a pH of 6.8 to 7.2.

[0052] In a preferred embodiment of the present invention, the parameters of the virus culture in step (B) include: a culture temperature of 35-37° C., a glucose concentration of 3-4 g / L in the culture medium, 40% dissolved oxygen, and a pH of 6.8-7.2.

[0053] In a preferred embodiment of the present invention, during the cell culture and virus culture process, the rotation speed of the reactor is 27-40 rpm.

[0054] In a preferred embodiment of the present invention, the virus titer in the virus suspension in step (B) is 8.5-10.0Log (TCID 50 / mL).

[0055] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0056] The following example uses a 50L cell reactor as an example to illustrate the culture method of porcine deltacoronavirus of the present application.

[0057] Example 1

[0058] A method for culturing porcine deltacoronavirus, the method comprising:

[0059] (1) Cell culture:

[0060] 1.1. ST cells (pig testicular cells) were inoculated in a 50L stirred cell reactor; the cell culture medium was serum-free SF601 medium, and the inoculation density of ST cells was 0.5-1.2×10 6 cells / mL, the cell culture volume is 16-20L;

[0061] 1.2. After ST cells were inoculated and cultured at 37°C for 3 days, serum-free SF601 medium was added to adjust the cell density to 2.0-3.5×10 6 cells / mL, and culture medium A was obtained, which was 0 hours of virus culture;

[0062] (2) Virus culture:

[0063] 2.1. Continue culturing medium A for 24 hours, add 5 μg / mL pancreatin (brand Sigma), and then add CaCl2·2H2O to the Ca content of the culture medium. 2+ The concentration is 0.5mmol / L;

[0064] 2.2. Then, the virus is inoculated, and the inoculation volume of the porcine delta coronavirus venom is 1.5% of the volume of the culture medium A; the temperature of the virus culture after inoculation is 35-37°C;

[0065] 2.3. Continue culturing for 24 hours after inoculation and add trypsin for the second time. The amount of trypsin added is 5 μg / mL. Continue culturing for 68 to 74 hours and harvest the virus suspension.

[0066] The harvest is when the virus titer is ≥8.5-10.0Log (TCID50 / mL) and the viability is 70-80%.

[0067] During the culture process of steps (1) and (2), 0.01-0.1 VVM of compressed air is introduced, and the dissolved oxygen and pH changes during the culture are adjusted by 0.01-0.1 VVM, the dissolved oxygen is 40%, the pH is 6.8-7.2, and when the pH is lower than 6.9, the reactor automatically controls the flow to add 0.9 mol / L sodium bicarbonate solution. At the same time, samples are taken every 24 hours to detect the density and viability of living cells, and the glucose and lactic acid contents are determined. When the glucose concentration is lower than 3 g / L, glucose is supplemented to 4 g / L.

[0068] Example 2

[0069] This embodiment is the same as Example 1 except that "the inoculation volume of porcine delta coronavirus venom in step 2.2 is 0.1% of the volume of culture medium A".

[0070] Example 3

[0071] This embodiment is the same as Example 1 except that "the inoculation volume of porcine deltacoronavirus venom in step 2.2 is 3% of the volume of culture medium A".

[0072] Example 4

[0073] In this example, except for step "1.1, the seeding density of ST cells is 1.5-1.8×10 6 Except for "cells / mL", the rest is the same as Example 1.

[0074] Example 5

[0075] In this example, except for step "1.1, the seeding density of ST cells is 0.1-0.3×106 Except for "cells / mL", the rest is the same as Example 1.

[0076] Example 6

[0077] In this embodiment, except for step "1.2, serum-free SF601 medium was added to adjust the cell density in the culture medium A to 1.5-1.8×10 6 Except for "cells / mL", the rest is the same as Example 1.

[0078] Example 7

[0079] In this embodiment, except for step "1.2, serum-free SF601 medium was added to adjust the cell density in the culture medium A to 3.8-4.3×10 6 Except for "cells / mL", the rest is the same as Example 1.

[0080] Example 8

[0081] This embodiment is the same as Example 1 except that in step 2.2, the temperature of virus culture after inoculation is 41°C.

[0082] Example 9

[0083] This embodiment is the same as Embodiment 1 except that in step 2.2, the temperature of virus culture after inoculation is 32°C.

[0084] Example 10

[0085] A method for culturing porcine deltacoronavirus, the method comprising:

[0086] (1) Cell culture: same as in Example 1;

[0087] (2) Virus culture:

[0088] 2.1. Continue culturing culture medium A for 24 hours and add 5 μg / mL pancreatin (brand: Sigma);

[0089] 2.2, same as Example 1;

[0090] 2.3, same as Example 1;

[0091] The process parameters in the cultivation process of steps (1) and (2) are the same as those in Example 1.

[0092] The virus culture stage of this comparative example does not include the step of adding calcium ions into the culture solution.

[0093] Embodiment 11

[0094] In this embodiment, except for step "2.1, the calcium ions are added in the form of CaCl2·2H2O; after the calcium ions are added, the Ca2+ The concentration of is 0 mmol / L. The rest is the same as in Example 1.

[0095] Example 12

[0096] In this embodiment, except for step "2.1, the calcium ions are added in the form of CaCl2·2H2O; after the calcium ions are added, the Ca 2+ The concentration of is 1 mmol / L. The rest is the same as in Example 1.

[0097] Embodiment 13

[0098] In this embodiment, except for step "2.1, the calcium ions are added in the form of CaCl2·2H2O; after the calcium ions are added, the Ca 2+ The concentration of is 2 mmol / L. The rest is the same as in Example 1.

[0099] Embodiment 14

[0100] In this embodiment, except for step "2.1, the calcium ions are added in the form of CaCl2·2H2O; after the calcium ions are added, the Ca 2+ The concentration of is 5 mmol / L. The rest is the same as in Example 1.

[0101] Comparative Example 1

[0102] A method for culturing porcine deltacoronavirus, the method comprising:

[0103] (1) Cell culture: Same as Example 1.

[0104] (2) Virus culture:

[0105] 2.1. Continue culturing medium A for 24 hours and add CaCl2·2H2O to the Ca 2+ The concentration is 0.5mmol / L;

[0106] 2.2, same as Example 1;

[0107] 2.3. Continue culturing for 68 to 74 hours after inoculation and harvest the virus suspension.

[0108] The process parameters in the cultivation process of steps (1) and (2) are the same as those in Example 1.

[0109] The virus culture stage of this comparative example does not include the step of adding trypsin.

[0110] Comparative Example 2

[0111] A method for culturing porcine deltacoronavirus, the method comprising:

[0112] (1) Cell culture: Same as Example 1.

[0113] (2) Virus culture:

[0114] 2.1. Same as Example 1;

[0115] 2.2, same as Example 1;

[0116] 2.3. Continue culturing for 68 to 74 hours after inoculation and harvest the virus suspension.

[0117] The process parameters in the cultivation process of steps (1) and (2) are the same as those in Example 1.

[0118] The virus culture stage of this comparative example does not include the step of adding trypsin for the second time.

[0119] Comparative Example 3

[0120] This comparative example is the same as Example 1 except that in step 2.2, the inoculation volume of porcine delta coronavirus venom is 3.3% of the volume of culture medium A.

[0121] Test Example 1

[0122] In order to show that the culture method of porcine deltacoronavirus of the present application can effectively improve the virus titer of porcine deltacoronavirus culture, the virus suspensions prepared in Examples 1 to 10 and Comparative Examples 1 to 4 were tested for virus titer, and the specific results are shown in Table 1:

[0123] Table 1:

[0124]

[0125]

[0126] As can be seen from the above, the virus suspensions prepared in Examples 1 to 3 of the present application have a higher virus titer, with a virus titer of 8.5 to 10.0 Log (TCID 50 / mL).

[0127] The seeding density of ST cells in Example 4 was 1.5-1.8×10 6 cells / mL. When the cell density was adjusted on the third day, the cells were in the stationary phase and could no longer meet the needs of virus culture.

[0128] In Example 5, the seeding density of ST cells was 0.1-0.3×10 6 cells / mL. When adjusting the cell density on the third day, there is no need to add fresh culture medium, as the nutrients in the old medium can no longer meet the needs of virus culture.

[0129] In Example 6, the cell density in the culture medium A after adjustment by adding serum-free SF601 culture medium in step 1.2 was 1.5-1.8×10 6 cells / mL, the cell density is low during virus culture, and the virus infects fewer host cells.

[0130] In Example 7, the cell density in the culture medium A after adjustment by adding serum-free SF601 medium in step 1.2 was 3.8-4.3×10 6 cells / mL, the density of virus culture cells is too high and the nutrients in the culture medium cannot meet the needs of virus culture.

[0131] Example 8 The temperature of virus culture after inoculation is 41°C. The activity of the virus will be inhibited or even cause the death of the virus under unsuitable environmental conditions, such as high temperature. Therefore, the temperature of 41°C is an unsuitable living condition for porcine coronavirus, and the virus cannot survive and replicate at such a temperature.

[0132] Example 9 The temperature of virus culture after inoculation is 32°C. The virus culture temperature is too low and the cells grow slowly.

[0133] The virus culture stage of Example 10 does not include the step of adding calcium ions to the culture medium. The virus titer of the virus obtained in this experimental example is significantly lower than that of Examples 1 to 3.

[0134] In the comparative example 1, the virus culture stage does not include the step of adding trypsin, and the comparative example 2 does not include the step of adding trypsin for the second time. The virus titer of the virus obtained in this experimental example is further reduced relative to the titer of Examples 1 to 3.

[0135] In comparative example 3, the inoculation volume of porcine delta coronavirus venom is 3.3% of the volume of culture medium A. The excessive addition of virus in this comparative example directly leads to cytotoxicity, which makes the cells unable to grow normally or die, thereby affecting the replication and amplification of the virus.

[0136] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing porcine deltacoronavirus, characterized in that: The culture method comprises: (A) Cell culture: culturing host cells to obtain culture medium A; (B) Virus culture: After culturing medium A for 24 hours, trypsin was added for the first time. Then, the virus was inoculated and cultured for another 24 hours. Trypsin was added for the second time. The virus suspension was harvested after culturing for 68-74 hours. During the inoculation process, the inoculation volume of porcine deltacoronavirus venom is 0.1-3% of the volume of culture medium A.

2. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The cell culture and virus culture are both carried out in a cell reactor; Preferably, the reactor is a 50L cell culture reactor or a 300L cell culture reactor.

3. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The culture medium in the cell culture process is serum-free SF601 culture medium; And / or, the culture medium in the virus infection culture process is serum-free SF601 culture medium.

4. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The host cell is a ST cell.

5. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: During the cell culture, the initial cell density of the host cells is: 0.5-1.2×10 6 cells / mL; When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.5 × 10 6 cells / mL.

6. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The amount of pancreatin added for the first time in step (B) is 5 mg / L; And / or, the amount of pancreatic enzyme added for the second time in step (B) is 5 mg / L.

7. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The virus culture process in step (B) also includes: adding calcium ions after adding pancreatic enzymes for the first time and before inoculating the virus.

8. The method for culturing porcine deltacoronavirus according to claim 7, characterized in that: The calcium ions are added in the form of CaCl2·2H2O; Preferably, after adding calcium ions, the Ca in the culture medium 2+ The concentration is 0.05~0.5mmol / L.

9. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The parameters of the cell culture in step (A) include: a culture temperature of 37° C., a glucose concentration of 3 to 4 g / L in the culture medium, 40% dissolved oxygen, and a pH of 6.8 to 7.2; And / or, the parameters of the virus culture in step (B) include: culture temperature 35-37° C., glucose concentration in the culture medium 3-4 g / L, dissolved oxygen 40%, pH 6.8-7.2; And / or, during the cell culture and virus culture process, the rotation speed of the reactor is 27-40 rpm.

10. The method for culturing porcine deltacoronavirus according to claim 1, characterized in that: The virus titer in the virus suspension in step (B) is 8.5-10.0 Log (TCID 50 / mL).

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