Method for improving yield of canine parvovirus vaccine strain

Abstract

The invention provides a method for increasing the yield of canine parvovirus vaccine strains. The method comprises the following steps: adding tunicamycin into a cell culture medium to culture and subculture MDCK cells, adding canine parvovirus, incubating and collecting cell supernatant. According to the invention, the yield of the canine parvovirus vaccine strain in cell production can be improved, and the production cost of the canine parvovirus vaccine is reduced. The canine parvovirus vaccine strain obtained by the invention is basically consistent with the existing canine parvovirus vaccine strain in the aspects of immunogenicity and protection provided after immunization, and has a huge application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology and immunology, and particularly relates to a method for improving the yield of a canine parvovirus vaccine strain. BACKGROUND

[0002] Canine parvovirus (CPV-2) is a kind of pathogen that seriously threatens canine animals, and the clinical manifestations of the infection in dogs are mainly hemorrhagic enteritis or myocarditis. The severity of the disease is closely related to the immune status, age and intervention after infection of the dog. At present, there is no specific drug for canine parvovirus disease, and the sick dogs often take supportive therapy such as fluid replacement and interferon, so the mortality rate is high. The prevention strategy through vaccination is one of the important measures to control canine parvovirus disease.

[0003] The production of canine parvovirus vaccine is mainly the amplification of the vaccine strain on cells, and the corresponding vaccine antigen solution is finally obtained by harvesting the supernatant of the culture. The suspension cell culture technology has become the preferred cell type for vaccine production due to the advantages of system closure, simple operation, stable culture and easy monitoring. At present, the only suspension cell used for the proliferation of canine parvovirus vaccine strain is MDCK cell (canine kidney cell), but due to the particularity of canine transferrin receptor type 1 (the main receptor of canine parvovirus), the proliferation level of canine parvovirus is limited to a certain extent, which affects the production efficiency and cost of the vaccine.

[0004] Transferrin receptor type 1 (TfR-1) is a homodimeric type II transmembrane protein on the cell surface, and its main function is to transport iron into the cell by binding and internalizing transferrin. The invasion of canine parvovirus into cells is mainly through the binding with canine cell surface TfR-1, and then mediating the endocytosis of host clathrin. There are certain differences in the amino acid homology of TfR-1 between different hosts, such as the homology of canine and cat TfR-1 is 88%, and these differences also determine the efficiency of virus invasion into different host cells. The relatively special point of canine TfR-1 is the presence of Asn at residue 384 in the sequence, which introduces a glycosylation site into the receptor domain. The added glycan not only blocks the binding of FPV and other parvoviruses with canine TfR-1, but also reduces the binding of CPV-2 with canine TfR-1 to a certain extent.

[0005] Therefore, by reducing the glycosylation of canine TfR-1 receptor and then enhancing the binding ability of CPV-2 with cells, the efficiency of virus invasion is enhanced. By using this characteristic, a new technical means is developed to improve the multiplication efficiency of canine parvovirus vaccine strain in MDCK cell culture production and increase the yield of vaccine. SUMMARY

[0006] Therefore, the application provides a method for improving the yield of canine parvovirus vaccine strains, which can reduce the production cost of the canine parvovirus vaccine and has great application prospect.

[0007] The technical scheme of the application is implemented as follows:

[0008] In a first aspect, the application provides a method for improving the yield of canine parvovirus vaccine strains, which comprises the following steps: adding tunicamycin to a cell culture medium to culture and subculture MDCK cells, adding canine parvovirus, incubating, and collecting the cell supernatant.

[0009] Tunicamycin has the function of inhibiting protein N-glycosylation, and its structural formula is as follows:

[0010]

[0011] Tunicamycin is added to the MDCK cell culture medium according to a fixed ratio for the whole process of cell recovery, culture expansion and virus proliferation. With the action of tunicamycin in MDCK cells, the adsorption and invasion efficiency of the virus is improved, and then the proliferation efficiency of the canine parvovirus on the cells is improved, and the yield of the vaccine strain is improved.

[0012] On the basis of the above technical scheme, further, the ratio of the amount of tunicamycin to the amount of MDCK cells is (0.02-10) μM: 1x10 5 Individuals / mL.

[0013] On the basis of the above technical scheme, further, the ratio of the amount of tunicamycin to the amount of MDCK cells is 2.5 μM: 1x10 5 Individuals / mL.

[0014] On the basis of the above technical scheme, further, the multiplicity of infection of the canine parvovirus is 0.1.

[0015] On the basis of the above technical scheme, further, the incubation is incubation in a 37℃, 30rpm, 5% CO2 incubator.

[0016] On the basis of the above technical scheme, further, the incubation time is 24h.

[0017] In a second aspect, the application provides a canine parvovirus vaccine strain obtained by the above method.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] (1) The application can improve the yield of canine parvovirus vaccine strains in cell production and reduce the production cost of the canine parvovirus vaccine.

[0020] (2) The canine parvovirus vaccine strain obtained by the present invention is basically consistent with the existing canine parvovirus vaccine strains in terms of immunogenicity and the protective effect provided after immunization, and has great application prospects. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The present invention provides a method for determining the effects of adding tunicamycin to a culture medium on cell growth rate and cytotoxicity. A represents the cell growth rate at different concentrations; B represents the determination of cytotoxicity at different concentrations.

[0023] Figure 2 To illustrate the effect of adding tunicamycin to the culture medium on increasing the yield of canine parvovirus vaccine strain provided by this invention, A represents the virus proliferation effect at different addition concentrations; B represents the effect of different addition methods on virus proliferation at the optimal addition concentration.

[0024] Figure 3 The present invention provides the effect of adding tunicamycin to the culture medium on virus adsorption and invasion; A shows the effect of adding tunicamycin on virus adsorption in cells; B shows the effect of adding tunicamycin on virus invasion in cells.

[0025] Figure 4 This invention provides a comparison of the immunogenicity of canine parvovirus vaccine strains produced by adding tunicamycin to a culture medium at a fixed ratio, with those produced using the original method. Detailed Implementation

[0026] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0027] In the following specific embodiments, unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] MDCK cells (canine kidney cells), tunicamycin, canine parvovirus (GenBank: MT183673.1).

[0029] In the following specific implementation, the cell growth medium and cell culture medium are both commercially available products.

[0030] Example 1: Detection of cell growth curve and activity of tunicamycin

[0031] 1. Detection of cell growth curves

[0032] Chlamydiaein was diluted with cell growth medium to 0 μM, 0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM, and 20 μM, and then added to the cell culture medium.

[0033] MDCK cells in good growth condition were passaged using the above-described cell culture medium. The cell density was adjusted to 1×10⁶ cells / year. 5 The cells were inoculated at a rate of 1 / mL into 125mL shake flasks.

[0034] The cells were incubated at 37℃, 30rpm, and 5%CO2. Samples were taken out at 24h, 48h, and 72h after incubation for cell counting and to calculate the cell growth rate.

[0035] Test results as follows Figure 1 As shown in Figure A, when the concentration of tunicamycin is less than 10 μM, the growth rate of suspended MDCK cells is not significantly affected.

[0036] 2. Cell viability detection

[0037] Chlamydiaein was diluted with cell growth medium to 0 μM, 0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM, 10 μM, and 20 μM, and then added to the cell culture medium.

[0038] MDCK cells in good growth condition were passaged using the above-mentioned cell culture medium, and the cell density was adjusted to 1×10⁶. 5 The number of cells / mL was inoculated into a 96-well plate, 100 μL per well.

[0039] Incubate at 37℃ in a 5% CO2 incubator for 48 hours.

[0040] Then remove the 96-well plate, discard the culture medium in the wells, wash three times with sterile PBS, and perform cell viability assay using the Cell Titer-One Solution Assay kit.

[0041] The 96-well plate was left to stand at room temperature for 30 min, 100 μL of CellTiter-Glo reagent was added to each well, and stirred on a shaker for 2 min, then the cells were incubated at room temperature in the dark for 10 min, and finally the luminescence signal was recorded with a multi-lane fluorescence reader.

[0042] The cell survival rate was determined to reflect the toxic effect of the compound on the cells, and the cell survival rate (%) was calculated according to the formula: cell survival rate (%) = average value of luminescence of cells treated with the compound / average value of luminescence of cell control group.

[0043] The detection results are shown in Figure 1 As shown in B, the toxicity of tunicamycin to MDCK cells was small at a concentration of less than 10 μM.

[0044] Example 2 Detection of the effect of tunicamycin on the proliferation of canine parvovirus vaccine strain

[0045] 1. Detection of the effect of tunicamycin treatment at different concentrations on the proliferation of canine parvovirus vaccine strain

[0046] Tunicamycin was diluted with cell growth solution to 0 μM, 0.02 μM, 0.1 μM, 0.5 μM, 2.5 μM and 10 μM, and added to the cell culture medium.

[0047] The well-grown suspension cells MDCK were passaged with the above cell culture medium. The cell density was adjusted to 1 x 10 5 The cells were inoculated in 125 mL shake flasks in a synchronous manner, and canine parvovirus vaccine strain (CPV-2) (MOI = 0.1) was added to each flask.

[0048] The flasks were placed in a 37°C, 30 rpm, 5% CO2 incubator, and after 24 hours of culture, the cell supernatant was collected and frozen in a -80°C refrigerator. The TCID 50 The titer of the progeny virus in the cell supernatant after 24 hours of culture was detected.

[0049] The detection results are shown in Figure 2 As shown in A, the titer of the progeny virus in the cell supernatant of the MDCK cells treated with tunicamycin was significantly higher than that of the group without tunicamycin treatment, and at a concentration of 2.5 μM, tunicamycin promoted the proliferation of CPV-2 best and the compound dosage was the least.

[0050] 2. Detection of the effect of tunicamycin at different addition modes on the proliferation of canine parvovirus vaccine strain

[0051] Tunicamycin was diluted with cell growth solution to 0 μM and 2.5 μM, and added to the cell culture medium.

[0052] The well-grown suspended cells MDCK cells were passaged with the above cell culture medium. The cell density was adjusted to 1 x 10 5 The well-grown suspended cells MDCK cells were passaged with the above cell culture medium. The cell density was adjusted to 1 x 10

[0053] Meanwhile, a control group was set, and the same concentration of tunicamycin was added at the time of infection. The well-grown suspended cells MDCK cells were passaged without adding tunicamycin in the cell culture medium. The cell density was adjusted to 1 x 10 5 The well-grown suspended cells MDCK cells were passaged with the above cell culture medium. The cell density was adjusted to 1 x 10

[0054] The cells were cultured in a 37℃, 30rpm, 5% CO2 incubator, and the cell supernatant was collected after 24, 48, 72, 96, 120, 144, and 168 hours of culture and stored in a -80℃ refrigerator. The TCID 50 The progeny virus titer of the virus liquid in the harvested cell supernatant was detected.

[0055] The detection results are shown in Table B. The progeny virus titer of the MDCK cells treated with 2.5 μM tunicamycin was significantly higher than that of the cells without tunicamycin treatment at each time point. Figure 2 B.

[0056] In addition, compared with the group in which the same concentration of tunicamycin was added at the time of infection, the group in which tunicamycin was added all the time had a better CPV-2 proliferation effect. According to the vaccine perfusion production process analysis, the group in which tunicamycin was added all the time could harvest the vaccine strain for 24 hours or more longer than the group in which tunicamycin was added at the time of infection.

[0057] Example 3: Effect of tunicamycin on adsorption and invasion of canine parvovirus

[0058] 1. Virus adsorption experiment

[0059] Tunicamycin was diluted to 2.5 μM and 0 μM with cell growth solution and added to the cell culture medium.

[0060] The suspended MDCK cells were cultured and passaged, and the cell density was adjusted to 1 x 10 5 The cells were inoculated in 12-well plates at 1 mL per well.

[0061] CPV-2 (MOI = 5) was added to each well, and the cells were incubated in a 4℃ refrigerator for 1 hour.

[0062] The cells were washed with DMEM three times, and the cell samples were collected and stored in a -80℃ refrigerator. The TCID50 The harvested virus was detected for titer.

[0063] The detection results are shown in Figure 3 A, and Figure 3 The virus adsorption experiment data of A show that the chitin increased the adsorption capacity of CPV-2 on the cells.

[0064] 2. Virus invasion experiment

[0065] The chitin was diluted with cell growth solution to 2.5 μM and 0 μM and added to the cell culture medium, and the suspended MDCK cells were cultured and passaged, and adjusted to a cell density of 1 x 10 5 / mL, and inoculated in 12-well plates at 1 mL per well.

[0066] CPV-2 (MOI = 5) was added to each well, and incubated in a 4°C refrigerator for 1 hour.

[0067] The cells were washed with DMEM three times, and new cell culture medium (containing chitin) was added, and the cell plate was incubated in a 37°C, 30 rpm, 5% CO2 incubator for 1 hour, and then the cells were collected and frozen in a -80°C refrigerator, and the TCID 50 The harvested virus was detected for titer.

[0068] As shown in Figure 3 B, the virus invasion experiment data show that the chitin increased the adsorption of CPV-2 on the cells, and the viruses adsorbed on the cells can effectively invade the cells.

[0069] Example 4 Comparison of immunogenicity of canine parvovirus vaccine antigen obtained after chitin treatment and vaccine antigen without chitin treatment

[0070] Ten-week-old healthy beagle dogs were randomly divided into three groups:

[0071] Non-immunized group: treated only with PBS;

[0072] Chitin vaccine group: the chitin concentration was 2.5 μM, and CPV-2 (MOI = 0.1) according to Example 2;

[0073] Pro-antigen group: the chitin concentration was 0 μM, and the rest was consistent with Example 2, as a positive control group;

[0074] The above three groups, each group of 5.

[0075] Subcutaneously inoculated with 10 6 TCID 50 of antigen, and immunized again after 21 days by the same route and at the same dose.

[0076] Blood was collected 21 days after the secondary immunization, serum was separated, and neutralizing antibodies against the vaccine strain after immunization were detected.

[0077] The detection results are shown in Table 1. Figure 4 As shown in Table 1, the activated neutralizing antibody levels of the two groups of beagles after immunization according to the normal process are consistent, which indicates that the antigen produced according to the application has no difference in immunogenicity with the antigen obtained by the original production method.

[0078] In summary, the application provides a method for improving the yield of canine parvovirus vaccine strains, and chitosan has excellent efficacy in promoting the adsorption of canine parvovirus into cells, and at a concentration without toxic effect on cells, the yield of canine parvovirus vaccine strains can be significantly improved. Chitosan can be combined with other methods for improving the yield of canine parvovirus vaccine strains to improve the production efficiency of canine parvovirus vaccine and reduce the production cost of the vaccine, and has great application prospect.

[0079] The above only describes the preferred embodiments of the application and is not intended to limit the application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the application shall be included in the protection scope of the application.

Claims

1. A method of increasing the yield of a canine parvovirus vaccine strain, characterized in that, It comprises the following steps: culturing and subculturing MDCK cells in cell culture medium with addition of lycogrise, adding canine parvovirus, incubating, and collecting cell supernatant.

2. The method for improving the production of canine parvovirus vaccine strain according to claim 1, characterized in that, The ratio of the use amount of the echinocandin and MDCK cells is (0.02-10) μM: 1 x 10 5 cells / mL.

3. The method for increasing the yield of canine parvovirus vaccine strain as described in claim 2, characterized in that, The ratio of the use of the chitosin and MDCK cells is 2.5 μM: 1 x 10 5 per mL.

4. The method for improving the production of canine parvovirus vaccine strain according to claim 1, wherein the culture medium is a medium containing 0.5-5% of a blood serum of a dog infected with canine parvovirus. The multiplicity of infection of the canine parvovirus is 0.

1.

5. The method for improving the production of canine parvovirus vaccine strain according to claim 1, wherein the culture medium is a medium containing 0.5-5% of a blood serum of a dog infected with canine parvovirus. The incubation is incubation in a 37℃, 30rpm, 5% CO2 incubator.

6. A canine parvovirus vaccine strain obtained by the method according to any one of claims 1 to 5.

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