Method for improving expression of bovine herpesvirus type 4 strain

Abstract

The invention discloses a method for improving bovine herpesvirus type 4 strain expression, and belongs to the technical field of virus and vaccine preparation. The method comprises the following steps that the bovine herpesvirus type 4 strain and a virus maintenance solution are mixed and then inoculated to seed virus production cells to be cultured, and the virus maintenance solution contains DMSO, trehalose, dexamethasone and IFN-gamma and can remarkably improve the expression titer of the bovine herpesvirus type 4 strain.

Description

Technical Field

[0001] This invention relates to the field of virus and vaccine preparation technology, specifically to a method for improving the expression of bovine herpesvirus type 4 strain. Background Technology

[0002] Animal husbandry is a crucial sector of livestock development, particularly beef and dairy cattle farming. In recent years, respiratory diseases in cattle have become increasingly prevalent in farms across various regions, posing a significant challenge to the development of my country's beef and dairy cattle industries. Bovine Respiratory Disease Syndrome (BRDC) is a major systemic disease in cattle, caused by a combination of factors including production and transportation, early weaning, climate, air quality, and husbandry practices. It can lead to diseases such as bovine pneumonia, transport fever, and bronchitis. One of the core pathogens of BRDC is bovine herpesvirus type 4 (BHV-4). BHV-4 is a major cause of bovine reproductive disorders, exhibiting strong affinity for vascular endothelial cells, mammary gland tissue, endometrium, and fetal tissue in dairy cows, potentially causing various diseases such as postpartum metritis and abortion. Because BHV-4 infection is often asymptomatic and can exacerbate clinical symptoms when co-infected with other pathogens, diagnosis and treatment are significantly challenging. Therefore, strengthening the health management of cattle herds and doing a good job in hygiene and disinfection, especially vaccinating cows before or in early pregnancy to enhance the immune protection of cows and fetuses, are effective measures to reduce the risk of BHV-4 transmission.

[0003] Currently, there are three main methods for preparing herpesvirus: virus culture and amplification, ultracentrifugation purification, and subunit vaccine preparation. Virus culture and amplification is the most classic and considered the "gold standard" method. It typically uses human embryonic lung fibroblasts (such as MRC-5 cells or Vero cells) as host cells. Virus samples are inoculated into a monolayer of cells, centrifuged to promote adsorption, and then incubated at 35-37°C. The virus is harvested after the cells exhibit significant cytopathic effect (CPE). Ultracentrifugation purification usually involves collecting infected cell culture medium, first centrifuging at low speed to remove cell debris, then ultracentrifuging to concentrate the virus particles, and finally purifying them to obtain high-purity virus particles. Subunit vaccine preparation involves genetically engineered cells to produce recombinant antigens through suspension culture. However, these methods suffer from high contamination risk, limited yield, difficulty in controlling CPE, cumbersome operation, impaired viral activity, and poor immunogenicity. Therefore, a more efficient method for preparing herpesvirus is still needed. Furthermore, herpesviruses are crucial for vaccine development. A safe and effective BHV-4 vaccine is the most economical method for controlling BHV-4 disease. However, there is currently no commercially available inactivated BHV-4 vaccine for this disease. Therefore, further research on the virus needed for vaccine development is necessary. BHV-4 is mostly a latent infection, and the clinical symptoms after mixed infection with other pathogens are diverse and complex. Diagnosis cannot be made solely based on clinical symptoms; we need to perform virus isolation and identification, serological diagnosis, and molecular biological testing. Currently, research on BHV-4 is limited. Therefore, screening a strain with good immunogenicity and high viral titer is of great significance for BHV-4 vaccine development and diagnostic method development. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention develops a virus maintenance solution and culture method that can effectively enhance the expression of bovine herpesvirus type 4 strain, significantly increasing virus titer and laying the foundation for the development of bovine herpesvirus type 4 vaccine.

[0005] This invention discloses a method for increasing the expression of bovine herpesvirus type 4 strain, comprising the following steps:

[0006] S1. Mix bovine herpesvirus type 4 strain with virus maintenance solution to obtain virus solution;

[0007] S2. Inoculate the viral fluid into virus production cells, and harvest the bovine herpesvirus type 4 strain after culturing.

[0008] The virus maintenance solution contains liquid virus culture medium, horse serum, dimethyl sulfoxide (DMSO), trehalose, dexamethasone, and IFN-γ.

[0009] Furthermore, the content of each component in the virus maintenance solution includes at least one of the following:

[0010] (1) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5-97.5% (v / v), the horse serum accounts for 2% (v / v), and the dimethyl sulfoxide accounts for 0.5-1.5% (v / v).

[0011] (2) The concentration of trehalose in the virus maintenance solution is 10-30 mM;

[0012] (3) The concentration of dexamethasone in the virus maintenance solution is 5-15 μM;

[0013] (4) The concentration of IFN-γ in the virus maintenance solution is 10-30 ng / mL.

[0014] Preferably, the content of each component in the virus maintenance solution is any one of the following:

[0015] (1) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 97% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1% (v / v); the concentration of trehalose is 20 mM; the concentration of dexamethasone is 10 μM; and the concentration of IFN-γ is 20 ng / mL.

[0016] (2) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 30 mM; the concentration of dexamethasone is 15 μM; and the concentration of IFN-γ is 30 ng / mL.

[0017] (3) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 20 mM; the concentration of dexamethasone is 15 μM; and the concentration of IFN-γ is 20 ng / mL.

[0018] (4) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 30 mM; the concentration of dexamethasone is 10 μM; and the concentration of IFN-γ is 30 ng / mL.

[0019] Furthermore, the liquid virus culture medium is DMEM liquid culture medium.

[0020] Furthermore, in step S2, the virus inoculation rate is 10-25% (v / v).

[0021] Furthermore, in step S2, the virus-producing cells are bovine kidney cells.

[0022] Furthermore, in step S2, the culture conditions are incubation at 36-38°C and 5% CO2.

[0023] A second objective of this invention is to provide the role of the bovine herpesvirus type 4 strain prepared by the method in the preparation of vaccines, specifically, using the prepared strain as an antigen.

[0024] Furthermore, the vaccine is an inactivated vaccine.

[0025] Furthermore, the vaccine is used to prevent or treat diseases caused by infection with bovine herpesvirus type 4.

[0026] A third objective of this invention is to provide a virus maintenance medium for culturing bovine herpesvirus type 4 strains, the virus maintenance medium comprising the following components:

[0027] Liquid culture medium;

[0028] Horse serum;

[0029] Dimethyl sulfoxide;

[0030] Trehalose;

[0031] Dexamethasone;

[0032] IFN-γ.

[0033] Further, based on a total volume percentage (v / v) of 100% for the liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid culture medium comprises 96.5–97.5% (v / v), horse serum comprises 2% (v / v), and dimethyl sulfoxide comprises 0.5–1.5% (v / v); trehalose comprises 10–30 mM; dexamethasone comprises 5–15 μM; and IFN-γ comprises 10–30 ng / mL.

[0034] More preferably, the content of each component in the virus maintenance solution is any one of the following:

[0035] (1) Based on a total volume percentage (v / v) of 100% for the liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid virus culture medium comprises 97% (v / v), and the horse serum comprises 2%.

[0036] The concentrations of dimethyl sulfoxide (DMSO) were 1% (v / v), trehalose was 20 mM, dexamethasone was 10 μM, and IFN-γ was 20 ng / mL.

[0037] (2) The total volume percentage of liquid virus culture medium, horse serum, and dimethyl sulfoxide is 100%.

[0038] The liquid virus culture medium comprised 96.5% (v / v) of the total volume, and horse serum comprised 2%.

[0039] The concentrations of dimethyl sulfoxide (DMSO) were 1.5% (v / v), trehalose was 30 mM, dexamethasone was 15 μM, and IFN-γ was 30 ng / mL.

[0040] (3) The total volume percentage of liquid virus culture medium, horse serum, and dimethyl sulfoxide is 100%.

[0041] The liquid virus culture medium comprised 96.5% (v / v) of the total volume, and horse serum comprised 2%.

[0042] The concentrations of dimethyl sulfoxide (DMSO) were 1.5% (v / v), trehalose was 20 mM, dexamethasone was 15 μM, and IFN-γ was 20 ng / mL.

[0043] (4) The total volume percentage of liquid virus culture medium, horse serum, and dimethyl sulfoxide is 100%.

[0044] The liquid virus culture medium comprised 96.5% (v / v) of the total volume, and horse serum comprised 2%.

[0045] The concentrations of dimethyl sulfoxide (DMSO) were 1.5% (v / v), trehalose was 30 mM, dexamethasone was 10 μM, and IFN-γ was 30 ng / mL.

[0046] The beneficial effects of this invention are:

[0047] This invention first screens suitable maintenance medium formulations for specific bovine herpesvirus subtypes. After ensuring that the maintenance medium does not alter viral characteristics and can improve the expression level of bovine herpesvirus type 4 strain, the dosage of each component in the maintenance medium is further optimized, ultimately achieving a yield of 10 strains after only 48-72 hours of virus culture. 7.1 TCID 50 / ml virus. Furthermore, this invention not only achieves enhanced expression of bovine herpesvirus type 4 strain, but also significantly increases the viral titer and production efficiency, providing a new option for vaccine preparation against bovine herpesvirus type 4 infection. Attached Figure Description

[0048] Figure 1 These are photographs of normal cells and diseased cells in Example 4. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0050] Example 1: Determination of Virus Maintenance Solution Formulation

[0051] 1. Cell Culture

[0052] MDBK cells (bovine kidney cells) that have grown into a dense monolayer after 2 days of culture were taken and digested with trypsin solution. The cell density was 1–2 × 10⁻⁶ cells / year. 5 Cells / ml were passaged in 6-well plates or cell flasks.

[0053] 2. Treatment of diseased materials:

[0054] Nasal swabs were collected from cattle exhibiting clinical respiratory symptoms and processed under low temperature conditions. The swabs were centrifuged at 10,000 rpm for 20 minutes at 4°C. The supernatant was collected and sterilized by filtration through a 0.22 μm filter membrane for later use.

[0055] 3. Virus culture

[0056] BHV-4 virus was passaged in densely monolayered MDBK cells for 72 hours, followed by blind passage. The prepared tissue virus solution was inoculated at 20% by volume and incubated at 36–38°C in a 5% CO2 incubator for 1–2 hours. The incubator was then discarded, and maintenance medium was added. The cells were incubated at 36–38°C in a 5% CO2 incubator for 72 hours before harvesting.

[0057] The different maintenance solutions and culture results are shown in Table 1-2.

[0058] Table 1 Composition of different maintenance solutions

[0059]

[0060]

[0061] Table 2. Virus culture results in different maintenance media

[0062] formula <![CDATA[Virus content (unit: log 10 TCID 50 / ml)]]> Formula 1 6.00 Formula 2 5.96 Formula 3 5.80 Formula 4 6.12 Formula 5 6.00 Formula 6 6.0 Formula 7 5.96 Formula 8 5.80 Formula 9 6.00 Formula 10 6.67 Formula 11 6.12

[0063] The results above indicate that formulation 10 is significantly better than the other groups, therefore, further optimization will be carried out based on this formulation.

[0064] Example 2: Optimization of the dosage of each component in the virus maintenance solution formulation

[0065] The virus culture steps are the same as in Example 1, except that the virus maintenance solution formulation is different. The composition of the virus maintenance solution for each group is shown in Table 3.

[0066] Table 3 Concentrations of each component in different virus maintenance fluid combinations

[0067]

[0068] The virus culture results are shown in Table 4.

[0069] Table 4. Virus culture results with different special maintenance solution combinations

[0070] formula <![CDATA[Virus content (unit: log 10 TCID 50 / ml)]]> Formula a 6.43 Formula b 6.96 Formula c 7.12 Formula d 6.43 Formula e 6.33 Formula f 6.20 Formula g 7.00 Formula h 6.33 Formula i 6.50 Formula j 6.43 Formula k 7.12

[0071] Example 3: Subculture

[0072] Using densely grown monolayer MDBK cells, ordinary maintenance medium (DMEM + 2% horse serum) and the maintenance medium of this invention (DMEM + 2% horse serum + 1% DMSO + 20 mmol / ml trehalose + 10 μmol / ml dexamethasone + 20 ng / mL IFN-γ) were added respectively. The prepared tissue virus solution was inoculated at a volume ratio of 10-25%. After incubation at 36-38°C in a 5% CO2 incubator for 1-2 hours, the solution was discarded, maintenance medium was added, and the cells were incubated at 36-38°C in a 5% CO2 incubator for 72 hours before harvesting.

[0073] Table 5 Results of Virus Content Determination in Passage of Five Viruses (Unit: log) 10 TCID 50 / ml)

[0074]

[0075] The results showed that when using densely grown monolayer MDBK cells for virus passage culture, after 72 hours of culture, cytopathic effects only appeared after blind passage to the P5th generation in the ordinary maintenance medium, while cytopathic effects appeared as early as the P3th generation in the maintenance medium of this invention. Cells exhibited phenomena such as cell shrinkage, exposed cytoplasm, and cell shedding. Furthermore, the virus content after the P5th generation was almost one titer higher than that in the ordinary maintenance medium.

[0076] Example 4: Identification of the virus

[0077] (1) Specificity

[0078] The virus was diluted to 200 TCID using serum-free DMEM. 50 0.1 ml of the neutralization solution was mixed with an equal volume of specific serum against bovine herpesvirus type 4 strain and incubated at 37°C for 1 hour. The neutralization solution was then inoculated until monoclonal antibodies were formed, and cytopathic effects were observed for 5–7 days. Results showed that no cytopathic effects were observed in the neutralization test group, while all cells in the virus control group exhibited cytopathic effects characterized by cell rounding. The cell control group showed no cytopathic effects (see results below). Figure 1 ).

[0079] (2) Immunofluorescence detection

[0080] The virus seed was diluted to 200 TCID with serum-free DMEM. 50 Mix 0.1 ml of DMEM with an equal volume of serum specific to bovine herpesvirus type 4 (BV4), incubate at 37°C for 1 hour, and then inoculate the neutralization solution into 10 wells of a 96-well cell culture plate that has grown into a monolayer of cells, 0.1 ml / well. Simultaneously, set up 10 wells each for virus control and healthy cell control, 10 wells for the virus control group, and inoculate the healthy cell control group with 0.1 ml of serum-free DMEM. Incubate the 96-well cell culture plates in a CO2 incubator at 36–38°C for 5–7 days. Wash 3 times with PBST. Fix with 80% pre-chilled acetone for 10 minutes, then wash 3 times with PBST. Add BV4 specific serum (1:300), incubate at 37°C for 1 hour, and wash 3 times with PBST. Add FITC secondary antibody (1:200), incubate at 37°C for 1 hour, and wash 3 times with PBST. Finally, add 50 μl of PBST to each well. Observation with an inverted fluorescence microscope showed that the virus reacted with positive serum with a specific antigen-antibody reaction. After the addition of the fluorescent secondary antibody, obvious specific fluorescence appeared in the field of view, while no fluorescence was observed in the blank control well.

[0081] (3) Toxicity to rabbits

[0082] The virus stock was diluted with physiological saline to a viral load of 10. 5.5 TCID 50 Five pregnant rabbits were inoculated with 1 ml of virus solution per vaginal cavity. After seven consecutive days of observation, three of the pregnant rabbits developed the disease after infection. Four days after infection, the rabbits developed vaginal congestion, inflammation, edema, and fever. One rabbit experienced fetal abortion.

[0083] (4) Immunogenicity

[0084] Ten healthy, susceptible calves aged 6-12 months were used. Five calves were injected intramuscularly with 2ml of vaccine (the inactivated vaccine solution, which passed sterility and virus inactivation tests, and had a virus content ≥10) via the neck. 7.0 TCID 50 / ml, add 206 adjuvant at a volume ratio of 1:1 to prepare bovine herpesvirus type 4 inactivated vaccine). Five cattle were not injected as a control. All were isolated and raised under the same conditions. 28 days post-immunization, blood samples were collected to test for bovine herpesvirus type 4 neutralizing antibodies. A neutralizing antibody titer ≥1:32 indicates a good immunization effect. The results are shown in the table below.

[0085] Table 6. Results of antibody level detection in bovine serum immunized with vaccine.

[0086]

[0087] (5) Virus purity test

[0088] Tests were conducted according to the appendix of the current Chinese Veterinary Pharmacopoeia (Volume III), and no bacterial, fungal, mycoplasma, or exogenous viral contamination was found. The results showed that all viruses were pure.

[0089] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A method for enhancing the expression of bovine herpesvirus type 4 strain, characterized in that, Includes the following steps: S1. Mix bovine herpesvirus type 4 strain with virus maintenance solution to obtain virus solution; S2. The viral fluid is inoculated into virus production cells, and after culture, the bovine herpesvirus type 4 strain is harvested. The virus maintenance solution contains liquid virus culture medium, horse serum, dimethyl sulfoxide, trehalose, dexamethasone, and IFN-γ.

2. The method according to claim 1, characterized in that, The virus maintenance solution contains at least one of the following components: (1) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5-97.5% (v / v), the horse serum accounts for 2% (v / v), and the dimethyl sulfoxide accounts for 0.5-1.5% (v / v). (2) The concentration of trehalose in the virus maintenance solution is 10-30 mM; (3) The concentration of dexamethasone in the virus maintenance solution is 5-15 μM; (4) The concentration of IFN-γ in the virus maintenance solution is 10-30 ng / mL.

3. The method according to claim 2, characterized in that, The content of each component in the virus maintenance solution is any one of the following: (1) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 97% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1% (v / v); the concentration of trehalose is 20 mM; the concentration of dexamethasone is 10 μM; and the concentration of IFN-γ is 20 ng / mL. (2) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 30 mM; the concentration of dexamethasone is 15 μM; and the concentration of IFN-γ is 30 ng / mL. (3) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), and dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 20 mM; the concentration of dexamethasone is 15 μM; and the concentration of IFN-γ is 20 ng / mL. (4) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), dimethyl sulfoxide accounts for 1.5% (v / v); the concentration of trehalose is 30 mM; the concentration of dexamethasone is 10 μM; and the concentration of IFN-γ is 30 ng / mL.

4. The method according to claim 1, characterized in that, The liquid virus culture medium is DMEM liquid culture medium.

5. The method according to claim 1, characterized in that, In step S2, the virus is inoculated at a volume ratio of 10-25%.

6. The method according to claim 1, characterized in that, In step S2, the virus-producing cells are bovine kidney cells.

7. The method according to claim 1, characterized in that, In step S2, the culture conditions are: incubation at 36-38℃ and 5% CO2.

8. The use of the bovine herpesvirus type 4 strain prepared by the method according to any one of claims 1-7 in the preparation of vaccines.

9. The application according to claim 8, characterized in that, The vaccine in question is an inactivated vaccine.

10. A virus maintenance solution, characterized in that, The virus maintenance medium, used for culturing bovine herpesvirus type 4 strain, consists of the following components: Liquid virus culture medium Horse serum, Dimethyl sulfoxide, Trehalose, Dexamethasone, IFN-γ; Preferably, based on a total volume percentage (v / v) of 100% for the liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid culture medium comprises 96.5–97.5% (v / v), the horse serum comprises 2% (v / v), and the dimethyl sulfoxide comprises 0.5–1.5% (v / v). Trehalose, concentration 10-30mM Dexamethasone, at a concentration of 5-15 μM, IFN-γ, concentration of 10-30 ng / mL; More preferably, the content of each component in the virus maintenance solution is any one of the following: (1) Based on a total volume percentage of 100% (v / v) for liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid virus culture medium comprised 97% (v / v), horse serum comprised 2% (v / v), dimethyl sulfoxide comprised 1% (v / v), trehalose comprised 20 mM, dexamethasone comprised 10 μM, and IFN-γ comprised 20 ng / mL. (2) Based on a total volume percentage (v / v) of 100% for liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid virus culture medium comprised 96.5% (v / v), horse serum comprised 2% (v / v), dimethyl sulfoxide comprised 1.5% (v / v), trehalose comprised 30 mM, dexamethasone comprised 15 μM, and IFN-γ comprised 30 ng / mL. (3) Based on a total volume percentage (v / v) of 100% for liquid virus culture medium, horse serum, and dimethyl sulfoxide, the liquid virus culture medium comprises 96.5% (v / v), horse serum comprises 2% (v / v), dimethyl sulfoxide comprises 1.5% (v / v), trehalose comprises 20 mM, dexamethasone comprises 15 μM, and IFN-γ comprises 20 ng / mL. (4) Based on the total volume percentage of liquid virus culture medium, horse serum and dimethyl sulfoxide as 100% (v / v), the liquid virus culture medium accounts for 96.5% (v / v), horse serum accounts for 2% (v / v), dimethyl sulfoxide accounts for 1.5% (v / v), trehalose concentration is 30 mM, dexamethasone concentration is 10 μM, and IFN-γ concentration is 30 ng / mL.

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