Preparation of a High-Efficiency Culture Medium for Promoting the Replication of Foot-and-Mouth Disease Virus

Abstract

The present invention belongs to the field of biotechnology, and particularly relates to the application of citronellol as a virus production enhancer and a culture medium containing citronellol. The present invention unexpectedly discovers that adding a certain amount of citronellol to the culture medium for culturing FMDV can significantly increase the replication level of FMDV, indicating that citronellol has a significant effect of promoting the replication of FMDV and can be used as an enhancer for the production of foot-and-mouth disease vaccines and for the production of FMDV or vaccines; moreover, the present invention discovers that the combination of aspartate, thiolactic acid and citronellol further significantly promotes the replication level of FMDV and is used for the high-efficient production of FMDV or vaccines; based on this, the present invention also provides a culture medium for promoting the production of FMDV or vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of citronellol as a virus production enhancer and a culture medium containing citronellol. Background Art

[0002] The Picornaviridae family is composed of the smallest group of RNA viruses, mainly including the genera Enterovirus, Rhinovirus, Cardiovirus, and Aphthovirus, etc. Among them, foot-and-mouth disease belongs to the genus Aphthovirus of the family Picornaviridae, and is an important disease that infects cloven-hoofed animals caused by foot-and-mouth disease virus.

[0003] Foot-and-mouth disease is an infectious disease common to cloven-hoofed animals (such as pigs, cows, sheep, camels, etc.) caused by foot-and-mouth disease virus (FMDV). FMDV belongs to the genus Aphthovirus of the family Picornaviridae. Currently, there are 7 serotypes, namely O, A, C, SAT1, SAT2, SAT3 (i.e., South African foot-and-mouth disease virus types 1, 2, and 3), and Asia1 (Asia type 1). There is almost no immune protection between different serotypes. Animals infected with one type of foot-and-mouth disease can still be infected with another type of foot-and-mouth disease virus and get sick.

[0004] Among them, the prevalence of type O is complex and the harm is the most serious. FMDV is prone to mutation, which is its natural property and the main reason for the long-term prevalence, difficult control, and purification of foot-and-mouth disease. Vaccination is currently recognized as the most important measure for preventing foot-and-mouth disease. Commercial foot-and-mouth disease vaccines are mainly virus inactivated vaccines, and their safety and effectiveness have been widely verified. The complete FMDV particles have a relative sedimentation coefficient of 146S after sucrose density gradient centrifugation. The 146S antigen content is a key quality control point in the production process of foot-and-mouth disease inactivated vaccines and a key indicator for evaluating vaccine quality. Currently, the production capacity of foot-and-mouth disease vaccine antigen is low and the cost is high, which has become one of the technical bottlenecks restricting the development of foot-and-mouth disease vaccines. Therefore, studying how to promote FMDV replication is beneficial to increasing the yield of vaccine antigen and providing new ideas for vaccine production.

[0005] Thiolactic acid, also known as 2-mercaptopropionic acid, is a colorless to light yellow liquid with a roasted meat-like aroma. It is an important organic synthesis raw material, mainly used in the preparation of herbicides, plastic heat stabilizers, and antioxidants. Citronellol is a colorless oily liquid with a special fresh rose-like aroma and a slightly bitter taste. It is a natural compound extracted from citronella and has functions and effects such as antibacterial, antiviral, antioxidant, anti-inflammatory, promoting digestion, and soothing muscles and the nervous system. Amino acids are a class of amphoteric organic compounds containing basic amino groups and acidic carboxyl groups. They are the basic building blocks of biological functional macromolecule proteins and are also essential substances for virus replication. The present invention unexpectedly found that adding a certain amount of aspartate, thiolactic acid, and citronellol to the culture medium for culturing FMDV can significantly increase the replication level of FMDV, indicating that these substances alone or in combination have a significant effect of promoting FMDV replication and can be used as a synergist for the production of foot-and-mouth disease vaccines for the efficient production of FMDV or vaccines. Summary of the Invention

[0006] The present invention provides an application of citronellol as a virus production synergist and a culture medium containing citronellol. Specifically, it includes the following content:

[0007] In the first aspect, the present invention provides an application of citronellol or a pharmaceutically acceptable salt thereof as a virus or virus vaccine production synergist, and the structural formula of the citronellol is shown as the following formula (Ⅰ):

[0008]

[0009] Preferably, the virus is a virus of the family Picornaviridae.

[0010] Preferably, the virus is foot-and-mouth disease virus.

[0011] Preferably, the citronellol is added with a pharmaceutically acceptable carrier and / or excipient to form any one of the dosage forms of powder injection, capsule, tablet, and suspension.

[0012] In the second aspect, the present invention provides a composition for promoting the production of a virus or a virus vaccine, and the composition includes citronellol, aspartic acid, and thiolactic acid; the structural formulas of the citronellol, aspartic acid, and thiolactic acid are shown as the following formulas (Ⅰ)-(Ⅲ) respectively:

[0013]

[0014] Preferably, the virus is a virus of the family Picornaviridae.

[0015] Preferably, the virus is foot-and-mouth disease virus.

[0016] Preferably, the composition is added with pharmaceutically acceptable carriers and / or excipients to form any dosage form of powder injection, capsules, tablets, and suspensions.

[0017] In a third aspect, the present invention provides an application of the composition described in the second aspect above in the preparation of a virus or a virus vaccine production synergist.

[0018] Preferably, the virus is a picornavirus.

[0019] Preferably, the virus is foot-and-mouth disease virus.

[0020] In a fourth aspect, the present invention provides a culture medium for increasing the antigen yield of a virus or a virus vaccine, and the culture medium is: based on RPMI-1640 medium, adding citronellol, aspartic acid, and thiodactic acid.

[0021] Preferably, the concentrations of citronellol, aspartic acid, and thiodactic acid in the culture medium are respectively: 20-40 mg / L, 50-200 mg / L, and 20-40 mg / L.

[0022] Preferably, the concentrations of citronellol, aspartic acid, and thiodactic acid in the culture medium are 40 mg / L, 100 mg / L, and 40 mg / L respectively.

[0023] Preferably, the virus is a picornavirus.

[0024] Preferably, the virus is foot-and-mouth disease virus.

[0025] In a fifth aspect, the present invention provides an application of the culture medium described in the fourth aspect above in the production of a virus or a vaccine.

[0026] Preferably, the virus is a picornavirus.

[0027] Preferably, the virus is foot-and-mouth disease virus.

[0028] The beneficial effects of the present invention are as follows: First, the present invention unexpectedly discovers that after adding citronellol, aspartic acid, and thiodactic acid to the culture medium for culturing FNDV, the replication level of FMDV can be significantly increased, indicating that citronellol, aspartic acid, and thiodactic acid respectively have the effect of promoting the replication of FMDV, and can be used as a synergist for FMDV virus expression or vaccine production, and for the production of FMDV virus or vaccine; Second, the present invention discovers that adding citronellol, aspartic acid, and thiodactic acid on the basis of RPMI-1640 medium can further significantly promote the replication level of FMDV. Based on this, the present invention provides a composition and a culture medium for promoting the replication of FMDV, realizing the efficient production of FMDV or vaccine. Description of the Drawings

[0029] Figure 1 Schematic diagram of aspartic acid promoting FMDV replication;

[0030] Figure 2 Schematic diagram of thiodactic acid promoting FMDV replication;

[0031] Figure 3 Schematic diagram of citronellol promoting FMDV replication;

[0032] Figure 4 Schematic diagram of aspartic acid, thiodactic acid and citronellol jointly promoting FMDV replication. Specific implementation mode

[0033] The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the following examples are all obtained by purchasing from conventional biochemical reagent companies unless otherwise specified.

[0034] Aspartic acid, thiodactic acid and citronellol are all purchased from sigma company.

[0035] Commercial RPMI-1640 is purchased from Thermo Fisher Scientific company.

[0036] The foot-and-mouth disease virus strain O / BY / CHA / 2010 was isolated by our team and stored in the Foot-and-Mouth Disease and Emerging Disease Epidemiology Team and the National Foot-and-Mouth Disease Reference Laboratory of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0037] Example 1 Evaluation experiment on the effect of aspartic acid promoting FMDV replication

[0038] 1. Preparation of medium containing aspartic acid

[0039] The content of aspartic acid in commercial RPMI-1640 medium is 20 mg / L; on the basis of commercial RPMI-1640 medium, a certain dose of aspartic acid is added respectively to increase its concentration to 50 mg / L, 100 mg / L and 200 mg / L.

[0040] 2. Preparation of samples of cells incubated with medium containing aspartic acid

[0041] PK-15 cells were incubated with the prepared medium containing aspartic acid, and then inoculated with FMDV O / BY / CHA / 2010 (0.5 MOI). The supernatant was collected 12 h after inoculation.

[0042] 3. Determination of FMDV titer

[0043] Determine the TCID of the supernatant obtained in the previous step 50, virus titer analysis was performed.

[0044] TCID 50 The measurement steps are as follows: Inoculate BHK-21 cells into 96-well plates 16 h in advance. After the cells form a monolayer, wash the BHK-21 cells 3 times with PBS, and inoculate the supernatant collected in step 2 (10 -1 -10 -10 ) as the infection blank well, and set up two columns of negative control wells. Add 100 μl of virus filtrate or serially diluted virus diluent to the infection wells, adsorb at 37 °C for 1 h, and gently shake once every 20 min to ensure uniform virus adsorption. After 1 h of adsorption, aspirate the supernatant and gently wash the plate once with PBS. Add virus maintenance medium. After 48 h, observe the cytopathic effect every 12 h. After 72 h, record the number of diseased wells and calculate the TCID 50 . Measure in parallel three times and take the average value as the final virus titer.

[0045] The experimental results are as Figure 1 shown. During the culture of BHK-21 cells, after adding aspartic acid to increase its dose to 50 mg / L, 100 mg / L, and 200 mg / L, the TCID 50 of FMDV increased significantly, indicating that with the increase in the dose of aspartic acid, the virus TCID 50 of FMDV was significantly increased, promoting the replication of FMDV.

[0046] Example 2 Evaluation experiment on the effect of thiolactic acid on promoting FMDV replication

[0047] 1. Preparation of medium containing thiolactic acid

[0048] On the basis of commercial RPMI-1640 medium, add different doses of thiolactic acid, and make its concentration 0 mg / L, 20 mg / L, and 40 mg / L respectively.

[0049] 2. Preparation of samples for incubating cells with medium containing thiolactic acid

[0050] Incubate PK-15 cells with the prepared medium containing thiolactic acid, and then inoculate FMDV O / BY / CHA / 2010 (0.5 MOI). Collect the supernatant 12 h after inoculation.

[0051] 3. Determination of FMDV titer

[0052] Measure the TCID 50 of the supernatant obtained in the previous step for virus titer analysis.

[0053] TCID 50 The measurement steps are the same as those in Example 1.

[0054] The experimental results are as follows Figure 2 shown. During the cultivation of BHK-21 cells, after adding thiodactic acid, the TCID 50 of FMDV increased significantly, indicating that the addition of thiodactic acid significantly increased the TCID 50 of FMDV and promoted the replication of FMDV. Example 3 Evaluation experiment on the effect of citronellol in promoting FMDV replication

[0055] 1. Preparation of medium containing citronellol

[0056] On the basis of commercial RPMI-1640 medium, different doses of citronellol were added respectively to make its concentrations 0 mg / L, 20 mg / L and 40 mg / L.

[0057] 2. Preparation of samples of cells incubated with medium containing citronellol

[0058] PK-15 cells were incubated with the prepared medium containing citronellol, and then inoculated with FMDV O / BY / CHA / 2010 (0.5 MOI). The supernatant was collected 12 h after inoculation.

[0059] 3. Determination of FMDV titer

[0060] The TCID 50 of the supernatant obtained in the previous step was measured for virus titer analysis.

[0061] The determination steps of TCID 50 are the same as those in Example 1.

[0062] The experimental results are as follows Figure 3 shown. During the cultivation of BHK-21 cells, after adding citronellol, the TCID 50 of FMDV increased significantly, indicating that the addition of citronellol significantly increased the virus TCID 50 of FMDV and promoted the replication of FMDV. Example 4 Evaluation experiment on the effect of aspartic acid, thiodactic acid and citronellol in jointly promoting FMDV replication

[0063] 1. Preparation of medium containing aspartic acid, thiodactic acid and citronellol

[0064] Based on commercial RPMI-1640 medium, a certain dose of aspartic acid, thiolactic acid, and citronellol was added, such that the concentrations of aspartic acid, thiolactic acid, and citronellol were 100 mg / L, 40 mg / L, and 40 mg / L respectively (Aspartate(100mg / L)+Thiolactic acid(40mg / L)+Citronellol(40mg / L)). Commercial RPMI-1640 medium was used as a control (Aspartate(20mg / L)+Thiolactic acid(0mg / L)+Citronellol(0mg / L)).

[0065] 2. Sample preparation of cells incubated with medium containing aspartic acid, thiolactic acid, and citronellol

[0066] PK-15 cells were incubated with the prepared medium containing aspartic acid, thiolactic acid, and citronellol, and then FMDV O / BY / CHA / 2010 (0.5 MOI) was inoculated. The supernatant was collected 12 h after inoculation.

[0067] 3. FMDV titer determination

[0068] The TCID of the supernatant obtained in the previous step was determined 50 , and virus titer analysis was performed.

[0069] TCID 50 The determination procedure was the same as in Example 1.

[0070] The experimental results were as Figure 4 shown. During the cultivation of BHK-21 cells, after simultaneously adding aspartic acid, thiolactic acid, and citronellol, the TCID 50 of FMDV increased significantly. Moreover, compared with adding aspartic acid, thiolactic acid, or citronellol alone, the TCID 50 increased significantly after simultaneously adding aspartic acid, thiolactic acid, and citronellol, indicating that aspartic acid, thiolactic acid, and citronellol could jointly increase the virus TCID 50 of FMDV, and the combined effect was better.

[0071] In summary, the present invention first discovers that after adding citronellol, aspartic acid, and thiosuccinic acid to the medium for culturing FMDV respectively, the replication level of FMDV can be significantly increased, indicating that citronellol, aspartic acid, and thiosuccinic acid respectively have the effect of promoting the replication of FMDV and can be used as synergists for FMDV virus expression or vaccine production, for the production of FMDV virus or vaccine; secondly, adding citronellol, aspartic acid, and thiosuccinic acid on the basis of RPMI-1640 medium can further significantly promote the replication level of FMDV. Based on this, the present invention provides a composition and a medium for promoting the replication of FMDV, realizing the efficient production of FMDV or vaccine.

[0072] It should be noted that adding citronellol, aspartic acid, and thiosuccinic acid to any host cell producing FMDV can promote the replication of FMDV virus, and thus promote the expression and production of FMDV virus.

[0073] It should be noted that Picornaviridae mainly includes the following four genera: Enterovirus, Rhinovirus, Cardiovirus, and Aphthovirus. Since the viral structural proteins among the four genera of Picornaviridae are highly conserved. Although the present invention takes FMDV of Picornaviridae virus as an example to prove that citronellol, aspartic acid, and thiosuccinic acid can promote the replication of FMDV virus, it is not limited to FMDV. On the basis that citronellol, aspartic acid, and thiosuccinic acid described in the present invention can promote the replication of FMDV virus, they can also promote the replication of other viruses of Picornaviridae.

Claims

1. The use of citronellol in the preparation of a foot-and-mouth disease virus vaccine production enhancer, characterized in that: The structural formula of the citronellol is shown in the following formula (I): 。 2. A composition for promoting the production of foot-and-mouth disease virus vaccine, characterized in that: The composition comprises citronellol, aspartic acid, and thiolactic acid; the structural formulas of the citronellol, aspartic acid, and thiolactic acid are respectively shown in the following formulas (I) to (III): 。 3. Use of the composition as claimed in claim 2 in the preparation of a synergist for the production of foot-and-mouth disease virus vaccine.

4. A culture medium for increasing the yield of foot-and-mouth disease virus vaccine antigen, characterized in that: The culture medium is: RPMI-1640 culture medium is used as the basic culture medium, and citronellol, aspartic acid and thiolactic acid are added.

5. The culture medium according to claim 4, characterized in that The concentrations of citronellol, aspartic acid and thiolactic acid in the culture medium are 20-40 mg / L, 50-200 mg / L and 20-40 mg / L respectively.

6. Use of the culture medium as described in any one of claims 4-5 in the production of foot-and-mouth disease virus vaccine.

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