Preparation method of porcine epidemic diarrhea virus and application of porcine epidemic diarrhea virus in improving titer and N protein expression of porcine epidemic diarrhea virus

Abstract

The invention provides a preparation method of a porcine epidemic diarrhea virus and application of the porcine epidemic diarrhea virus in improving the titer and N protein expression of the porcine epidemic diarrhea virus, and relates to the technical field of biology. The preparation method of the porcine epidemic diarrhea virus provided by the invention comprises the following steps: inoculating host cells with the porcine epidemic diarrhea virus, adding calcium salt in the virus culture process to regulate the concentration of calcium ions in a culture medium to 0.6-5.1 mmol / L, and continuously culturing to obtain the porcine epidemic diarrhea virus. The preparation method is simple in process, and the cultured porcine epidemic diarrhea virus is high in titer and high in N protein expression quantity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing porcine epidemic diarrhea virus and its application in improving porcine epidemic diarrhea virus titer and N protein expression. Background Technology

[0002] Porcine epidemic diarrhea virus (PEDV) is a major pathogen causing viral diarrhea in pigs. PEDV can infect pigs of almost all ages, with acute enteritis, vomiting, watery diarrhea, and dehydration as the main clinical symptoms. The morbidity and mortality rate of newborn piglets under 7 days old can be as high as 100%, causing serious economic losses to the pig industry.

[0003] PEDV is a member of the genus Alpha-coronavirus, approximately 28 kb in length, encoding four structural proteins (S, M, E, and N proteins), 16 non-structural proteins (nsp1–nsp16), and one accessory protein (ORF3). The N protein is a nucleocapsid protein that primarily plays a role in evading the host's innate immunity. The N gene sequence is highly conserved across different strains. Early PEDV infection produces large amounts of N protein antibodies; therefore, the N protein is considered to play a crucial role in inducing cell-mediated immunity. Due to these characteristics, the N protein is commonly used as a target for diagnostics and vaccine development, and can also be used as a detection item in the manufacturing process of inactivated virus vaccines.

[0004] Regarding virus inoculation processes, traditional PED viral vaccine production involves growing ST cells (porcine testicular cells) to a certain density, then changing the medium before inoculating with PED virus or directly inoculating with PED virus. However, primary virus cultures often result in insufficient virus titers and low N protein expression levels, posing a challenge for vaccine development. Current technologies use CDST medium to culture ST cells and inoculate them with PED virus for expression. Improving PED virus expression primarily involves adjusting cell culture parameters and optimizing culture control parameters, such as rotation speed, pH, temperature during the virus expression phase, and osmotic pressure. However, these methods have limited effectiveness in enhancing PED virus N protein expression.

[0005] Studies have found that intracellular calcium ion concentration increases significantly after viral infection of cells. Since calcium ions are important intracellular signaling molecules, changes in cellular activity are often accompanied by changes in calcium ion concentration. Research indicates that calcium-regulated signaling pathways play a crucial role in viral production. These pathways can translate various extracellular signals into diverse cellular responses, regulate viral replication and spread, promote mRNA precursor processing and translation, thereby improving the translation efficiency of viral proteins.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The primary objective of this invention is to provide a method for preparing porcine epidemic diarrhea virus to solve the aforementioned problems.

[0008] A second objective of this invention is to provide the application of the above-described preparation method in increasing the titer of porcine epidemic diarrhea virus or the expression of N protein.

[0009] To achieve the above objectives, the following technical solution is proposed:

[0010] In a first aspect, the present invention provides a method for preparing porcine epidemic diarrhea virus, wherein porcine epidemic diarrhea virus is inoculated into host cells, calcium salt is added during virus culture to adjust the calcium ion concentration in the culture medium to 0.6-5.1 mmol / L, and porcine epidemic diarrhea virus is obtained after continued culture.

[0011] As a further technical solution, the host cell includes ST cells;

[0012] Preferably, the viable cell density of the host cells is 2.0 × 10⁻⁶. 6 ~4.0×10 6 cells / mL, preferably 3.0 × 10⁻⁶. 6 cells / mL.

[0013] As a further technical solution, the inoculation amount of the virus is 0.03 to 0.1 MOI, preferably 0.065 MOI.

[0014] As a further technical solution, calcium salts are added to regulate the calcium ion concentration in the culture when porcine epidemic diarrhea virus is inoculated into host cells;

[0015] Preferably, the calcium salt comprises calcium chloride;

[0016] Preferably, the culture medium includes serum-free CD ST medium, and more preferably CD ST 258 medium.

[0017] As a further technical solution, the virus culture process also includes a step of adding trypsin;

[0018] Preferably, trypsin is added at 20–28 h and 44–52 h during the expression phase of porcine epidemic diarrhea virus, with a final concentration of 1–10 mg / L each time, preferably 5 mg / L.

[0019] As a further technical solution, the virus is harvested after culturing for 54–96 hours during the virus expression stage, with the preferred culturing time being 72 hours.

[0020] As a further technical solution, a host cell culture step is included before inoculation with porcine epidemic diarrhea virus, including: inoculating host cells into a culture medium for culture, adding culture medium to dilute the host cells after culture, and then inoculating with porcine epidemic diarrhea virus.

[0021] As a further technical solution, the seeding density of the host cells is 0.50 × 10⁻⁶. 6 ~2.0×10 6 cells / mL, preferably 1.0 × 10⁻⁶. 6 cells / mL;

[0022] Preferably, the culture time of the host cells is 60-84 hours, and more preferably 72 hours.

[0023] As a further technical solution, the infection time (TOI) of the virus is 22-26 hours, preferably 24 hours.

[0024] Secondly, the present invention provides the application of the above preparation method in improving the titer of porcine epidemic diarrhea virus or the expression of N protein.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The inventors discovered that adjusting the calcium ion concentration in the culture medium to 0.6–5.1 mmol / L during the culture of porcine epidemic diarrhea virus (PEDV) can significantly increase the titer of PEDV or the expression of N protein. The method for preparing PEDV provided by this invention is simple and yields PEDV with high titers and high N protein expression levels. Detailed Implementation

[0027] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0028] It should be noted that specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many ways other than those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] The embodiments and examples of the present invention will be described in detail below with reference to the implementation methods and examples. However, those skilled in the art will understand that the following implementation methods and examples are only for illustrating 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 skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In a first aspect, the present invention provides a method for preparing porcine epidemic diarrhea virus, wherein porcine epidemic diarrhea virus is inoculated into host cells, calcium salt is added during virus culture to adjust the calcium ion concentration in the culture medium to 0.6-5.1 mmol / L, and porcine epidemic diarrhea virus is obtained after continued culture.

[0031] In this invention, after adding calcium salt, the concentration of calcium ions in the culture medium can be, for example, but not limited to, 0.6 mmol / L, 0.7 mmol / L, 0.8 mmol / L, 0.9 mmol / L, 1.0 mmol / L, 2.0 mmol / L, 3.0 mmol / L, 4.0 mmol / L or 5.1 mmol / L, preferably 0.6 to 2.1 mmol / L.

[0032] The inventors discovered that when the calcium ion concentration is controlled between 0.6 and 2.1 mmol / L, the viral titer can be increased from 7.40 Log(TCID) to 100 mmol / L using the existing PED virus expression process. 50 The value was increased to 8.00 Log(TCID / mL). 50 The expression level of N protein increased from 20ug / mL to over 60ug / mL, which greatly improved the viral titer and N protein content of PED antigen or seed virus at harvest.

[0033] The method for preparing porcine epidemic diarrhea virus of the present invention is simple. By increasing the concentration of calcium ions in the culture medium during inoculation, the viral yield and N protein expression are improved. Finally, the appropriate calcium ion addition time and concentration range are screened to achieve a significant increase in PED virus titer and N protein expression.

[0034] In some alternative implementations, the host cell is an ST cell;

[0035] Preferably, the viable cell density of the host cells can be, for example, but is not limited to, 2.0 × 10⁻⁶. 6 cells / mL, 3.0×10 6 cells / mL or 4.0 × 10 6 cells / mL, preferably 3.0 × 10⁻⁶. 6 cells / mL.

[0036] In some alternative implementations, the amount of virus inoculated may be, for example, but not limited to, 0.03 MOI, 0.04 MOI, 0.05 MOI, 0.06 MOI, 0.07 MOI, 0.08 MOI, 0.09 MOI or 0.1 MOI, preferably 0.065 MOI.

[0037] In some alternative implementations, calcium salts are added to adjust the calcium ion concentration in the culture when porcine epidemic diarrhea virus is inoculated into host cells;

[0038] Preferably, the calcium salt comprises calcium chloride;

[0039] Preferably, the culture medium includes serum-free CD ST medium, and more preferably CD ST 258 medium.

[0040] In some alternative implementations, the virus culture process also includes a step of adding trypsin;

[0041] Preferably, trypsin is added at 20–28 h and 44–52 h during the expression phase of porcine epidemic diarrhea virus. The final concentration of trypsin added each time can be, for example, but not limited to, 1 mg / L, 3 mg / L, 5 mg / L, 7 mg / L, or 10 mg / L, but preferably 5 mg / L.

[0042] It should be noted that the "virus expression stage" in this invention refers to the stage from the adjustment period of inoculating host cells to the logarithmic phase of virus inoculation, and then to the harvesting of the virus. The virus expression stage 0h is when the host cells are inoculated.

[0043] In some optional embodiments, the virus is harvested after culturing for 54–96 hours during the virus expression phase. In this invention, the virus culture time can be, for example, but not limited to, 54h, 60h, 66h, 72h, 78h, 84h, 90h, 96h, etc., preferably 72h.

[0044] By optimizing and adjusting the various parameters in the preparation method of this invention, the viral titer and N protein expression level of the prepared virus are higher.

[0045] In some alternative implementations, a host cell culture step is included before inoculation with porcine epidemic diarrhea virus, including: inoculating host cells in a culture medium for culture, adding culture medium to dilute the host cells after culture, and then inoculating with porcine epidemic diarrhea virus.

[0046] In some alternative embodiments, the seeding density of the host cells may be, for example, but not limited to, 0.5 × 10⁻⁶. 6 cells / mL, 0.6×10 6 cells / mL, 0.7×10 6cells / mL, 0.8×10 6 cells / mL, 0.9×10 6 cells / mL, 1.0×10 6 cells / mL or 2.0 × 10 6 cells / mL, preferably 1.0 × 10⁻⁶. 6 cells / mL;

[0047] Preferably, the culture time of the host cells can be, for example, but not limited to, 60h, 62h, 64h, 66h, 68h, 70h, 72h, 74h, 76h, 78h, 80h, 82h, 84h, with 72h being the most preferred.

[0048] In some alternative implementations, the infection time of the virus can be, for example, but not limited to, 22h, 23h, 24h, 25h or 26h, preferably 24h.

[0049] Secondly, the present invention provides the application of the above preparation method in improving the titer of porcine epidemic diarrhea virus or the expression of N protein.

[0050] The method for preparing porcine epidemic diarrhea virus provided by this invention is simple and yields porcine epidemic diarrhea virus with high titer and high N protein expression. Therefore, the preparation method of this invention can be used to improve the titer or N protein expression of porcine epidemic diarrhea virus.

[0051] The present invention will be further illustrated below with specific embodiments and comparative examples. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0052] Preparation Example

[0053] This preparation example provides culture media for ST cells with different CaCl2·2H2O concentrations (the original culture medium contained approximately 0.1 mmol / L of calcium ions). A 100 mmol / L CaCl2·2H2O stock solution was prepared using the CaCl2·2H2O reagent as a calcium ion addition reagent. The required amount of stock solution to add for different concentrations of calcium ions in the CD ST 258 culture medium is shown in Table 1.

[0054] Table 1

[0055]

[0056] The following is a solution of 100 mmol / L CaCl2·2H2O:

[0057] To prepare 1 L of 100 mmol / L CaCl2·2H2O solution, according to the formula for calculating solution concentration, C = m / V, 100 mmol / L = m / 1L. That is, to prepare 1 L of 100 mmol / L CaCl2·2H2O solution, 0.1 mol of CaCl2·2H2O reagent is needed. Based on the molecular weight of CaCl2·2H2O being 147, 14.7 g of CaCl2·2H2O reagent needs to be weighed out.

[0058] Where C represents the concentration of the solution, in mol / L (mol / L) or g / L (g / L); m represents the mass of the required solute, in mol (mol) or g (g); and V represents the volume of the solution, in L (L).

[0059] Example 1

[0060] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0061] (1) ST cell culture: at 1.0×10 6 Cells were seeded in CD ST 258 medium and cultured at 37°C for 72 h, with cell counts performed daily.

[0062] (2) Adjusting ST cell density: Based on the viable cell density at 72 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0063] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of TOI 24h and 0.065 MOI, and the culture temperature was adjusted to 35℃.

[0064] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA. The PED virus was harvested at 96h. Three treatment groups were set up. 0.5mmol / L CaCl2·2H2O was added to the culture medium at 0h, 24h and 48h during the virus expression stage, respectively. 5mg / L trypsin was added to the culture medium in each treatment group at 24h and 48h during the virus expression stage.

[0065] Example 2

[0066] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0067] (1) ST cell culture: at 1.0×106 Cells were seeded in CD ST 258 medium and cultured at 37°C for 72 h, with cell counts performed daily.

[0068] (2) Adjusting ST cell density: Based on the viable cell density at 72 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0069] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of TOI 24h and 0.065 MOI, and the culture temperature was adjusted to 35℃.

[0070] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA. The PED virus was harvested at 96h. Three treatment groups were set up. 1 mmol / L CaCl2·2H2O was added to the culture medium at 0h, 24h and 48h during the virus expression stage, respectively. 5 mg / L trypsin was added to the culture medium in each treatment group at 24h and 48h during the virus expression stage.

[0071] Example 3

[0072] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0073] (1) ST cell culture: at 1.0×10 6 Cells were seeded in CD ST 258 medium and cultured at 37°C for 72 h, with cell counts performed daily.

[0074] (2) Adjusting ST cell density: Based on the viable cell density at 72 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0075] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of TOI 24h and 0.065 MOI, and the culture temperature was adjusted to 35℃.

[0076] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA and the osmotic pressure of the culture medium was detected by an ice point osmometer. The PED virus was harvested at 96h during the virus expression stage. Three treatment groups were set up, and 2mmol / L CaCl2·2H2O was added at 0h, 24h and 48h during the virus expression stage, respectively. 5mg / L trypsin was added to the culture medium in each treatment group at 24h and 48h during the virus expression stage.

[0077] Example 4

[0078] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0079] (1) ST cell culture: at 1.0×10 6 Cells were seeded in CD ST 258 medium and cultured at 37°C for 72 h, with cell counts performed daily.

[0080] (2) Adjusting ST cell density: Based on the viable cell density at 72 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0081] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of TOI 24h and 0.065 MOI, and the culture temperature was adjusted to 35℃.

[0082] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA and the osmotic pressure of the culture medium was detected by an ice point osmometer. The PED virus was harvested at 96h during the virus expression stage. Three treatment groups were set up, and 5 mmol / L CaCl2·2H2O was added to the culture medium at 0h, 24h and 48h during the virus expression stage, respectively. 5 mg / L trypsin was added to the culture medium in each treatment group at 24h and 48h during the virus expression stage.

[0083] Example 5

[0084] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0085] (1) ST cell culture: at 0.5×10 6 Cells were seeded in CD ST 258 medium and cultured at 37°C for 84 hours, with cell counts performed daily.

[0086] (2) Adjusting ST cell density: Based on the viable cell density at 84 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 2.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0087] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of TOI 22h and 0.03MOI, and the culture temperature was adjusted to 35℃.

[0088] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA. The PED virus was harvested at 96h. 0.5mmol / L CaCl2·2H2O was added to the culture medium at 28h during the virus expression stage. 10mg / L trypsin was added to the culture medium at 28h and 52h during the virus expression stage.

[0089] Example 6

[0090] This embodiment provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0091] (1) ST cell culture: at 2.0×10 6 Cells were seeded in CD ST 258 medium and cultured at 37°C for 60 h, with cell counts performed daily.

[0092] (2) Adjusting ST cell density: Based on the viable cell density at 60h of ST cell culture, fresh culture medium was added to adjust the cell density to 4.0×10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0093] (3) Virus inoculation: The virus (isolated strain XJ-HM, KM386647.1) was inoculated into the culture medium containing ST cells at an inoculation amount of 0.1 MOI 26h and 0.1 MOI, and the culture temperature was adjusted to 35℃.

[0094] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA. The PED virus was harvested at 96h. 0.5mmol / L calcium nitrate was added to the culture medium at 20h during the virus expression stage. 5mg / L trypsin was added to the culture medium at 20h and 44h during the virus expression stage.

[0095] Comparative Example 1

[0096] This comparative example provides a method for preparing porcine epidemic diarrhea virus, the method comprising the following steps:

[0097] (1) ST cells were cultured in CD ST 258 medium and inoculated with PED virus for PED virus expression. During the ST cell culture phase: 1.0 × 10⁶ cells / year were cultured in medium. 6 Cells were seeded at 100 cells / mL, cultured for 72 hours, and cell counts were performed daily.

[0098] (2) Adjusting ST cell density: Based on the viable cell density at 72 hours of ST cell culture, fresh culture medium was added to adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 cells / mL, this is recorded as the virus expression stage 0h;

[0099] (3) Virus inoculation: The virus was inoculated into the culture medium containing ST cells at an inoculation rate of TOI 24h and 0.065 MOI, and the culture temperature was adjusted to 35℃.

[0100] (4) Culture: Cells were sampled and counted at 24h, 42h, 48h, 72h and 96h during the virus expression stage. The biochemical parameters of the cell culture medium were detected by NOVA and the osmotic pressure of the culture medium was detected by an ice point osmometer. At 24h and 48h during the virus expression stage, 5mg / L trypsin was added to the culture medium. The PED virus was harvested at 96h during the expression stage.

[0101] Comparative Example 2

[0102] The difference from Example 1 is that in step (4), 8 mmol / L of CaCl2·2H2O is added after 24 hours of virus expression;

[0103] Test Example 1

[0104] Test samples: PED virus expression methods provided in Examples 1-6, PED virus expression methods provided in Comparative Examples 1-2, and samples from the positive control group during the expression process of 48-96 hours;

[0105] Test items: Viral titer of samples during the 48-96h expression process;

[0106] The positive control group was prepared by: [cell seeding density PCD 3.0 × 10⁶]. 6 Inoculation with the virus at a concentration of cells / mL, TOI 0h, and MOI 0.065 was performed in the above-mentioned medium containing ST cells. The culture temperature was adjusted to 35℃, and the virus was harvested after 72 hours. Reactor culture parameters: Taking a 10L reactor as an example: the culture speed was 70-100 rpm, the pH of the medium was 7.0-7.2, and the temperature for the virus expression stage was 35℃.

[0107] The details are shown in Table 2 below:

[0108] Table 2

[0109]

[0110] Note: Taking "0.5-0h" as an example, the meaning in parentheses in the first column of the table is that 0.5 mmol / L of CaCl2·2H2O is added to the culture medium when the virus expression is 0h.

[0111] As shown in Table 2, the present invention can improve the viral titer and N protein expression by adding calcium ions to the cell culture medium for an additional 24 hours during the viral expression stage.

[0112] Among them, when calcium ions were added at a concentration of 0.5 mmol / L during the 24-hour viral expression stage, the viral titer and N protein expression level at each time point after inoculation were significantly increased compared with the viral titer and N protein expression level of Comparative Example 1 and the positive control group.

[0113] In particular, when the culture time after PED virus inoculation reaches 72 h, and an additional 0.5 mmol / L calcium ion concentration is added during the 24 h virus expression phase, and 5 mg / L trypsin is added during the 24 h and 48 h virus expression phases, the viral titer of the expressed virus can be increased from 7.4 Log(TCID) to the original PED virus expression process (Comparative Example 1). 50 / mL) increased by 8.54 Log(TCID) 50 The N protein level increased from 20–40 μg / mL to 120 μg / mL. This significantly increased the N protein expression level of PED antigen or seed virus at harvest.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 of preparing a porcine epidemic diarrhea virus, characterized by, The porcine epidemic diarrhea virus is inoculated into host cells, and a calcium salt is added to regulate the calcium ion concentration in the culture medium to 0.6-5.1 mmol / L during the virus culture process, and the porcine epidemic diarrhea virus is prepared after further culture.

2. The production method according to claim 1, characterized by, The host cells include ST cells. Preferably, the viable cell density of the host cell is 2.0 x 10 6 cells / mL, preferably 3.0 x 10 6 cells / mL, and more preferably 4.0 x 10 6 cells / mL.

3. The preparation method according to claim 1, characterized in that, The inoculation amount of the virus is 0.03-0.1 MOI, preferably 0.065 MOI.

4. The method of claim 1, wherein, The calcium salt is added to regulate the calcium ion concentration in the culture medium during the inoculation of the porcine epidemic diarrhea virus into host cells. Preferably, the calcium salt includes calcium chloride. Preferably, the culture medium includes serum-free CD ST culture medium, preferably CD ST 258 culture medium.

5. The method of claim 1, wherein, The virus culture process further includes a step of adding trypsin. Preferably, the trypsin is added at 20-28 h and 44-52 h of the expression stage of the porcine epidemic diarrhea virus, and the final concentration of the trypsin is 1-10 mg / L, preferably 5 mg / L.

6. The method of claim 1, wherein, The virus is harvested after 54-96 h of the virus expression stage, preferably 72 h.

7. The method of any one of claims 1-6, wherein, The virus inoculation process further includes a step of culturing host cells, which includes inoculating host cells into a culture medium for culture, diluting the host cells with the culture medium after the culture, and then inoculating the porcine epidemic diarrhea virus.

8. The preparation method according to claim 7, characterized in that, The seeding density of the host cells is 0.50 x 10 6 ~ 2.0 x 10 6 cells / mL, preferably 1.0 x 10 6 cells / mL; Preferably, the culture time of the host cells is 60-84 h, preferably 72 h.

9. The preparation method according to claim 7, characterized in that, The infection time of the virus is 22-26 h, preferably 24 h.

10. Use of the preparation method in any one of claims 1-9 in improving the titer of porcine epidemic diarrhea virus or the expression of N protein.

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