A cell culture medium and culture method for promoting proliferation of porcine epidemic diarrhea virus

Abstract

The present invention relates to improvements in cell culture. The present invention describes improved methods and compositions for the production of porcine epidemic diarrhea virus (PEDV) antigens in mammalian cell culture. In addition, the present invention provides improved cell culture media, including improved production media, feed solutions, and combination feeds, which can be used to increase the efficiency of mammalian cell PEDV propagation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological cell culture, and particularly relates to a method for improving a proliferation culture medium. BACKGROUND

[0002] Cell vaccine is the leading direction of future animal vaccine research and application. How to improve virus titer in a limited number of cells and reduce cost is one of the key technical bottlenecks restricting animal vaccine production. In the process of animal vaccine production, optimization of cell culture virus scheme is an important factor.

[0003] Viruses can edit the metabolic pathways of host energy production to provide more energy and biosynthetic resources to drive viral protein synthesis and produce infectious particles. Therefore, activators of metabolic pathways are likely to improve the ability of viruses to infect host cells, thereby facilitating efficient proliferation of viruses in cells. Therefore, by adding metabolic substances in the cell line for culturing viruses, virus proliferation is promoted, the maximum virus harvest is obtained in the same culture space, the effective antigen amount for subsequent vaccine preparation is increased, and the immune effect is enhanced.

[0004] Porcine epidemic diarrhea (PED) is an acute, highly contagious enteric disease of pigs caused by porcine epidemic diarrhea virus (PEDV). PED is very harmful to pigs, and is most harmful to suckling pigs. Since 2010, PED has broken out on a large scale in pig farms in most parts of the country, with an estimated hundreds of millions of piglets dying nationwide, causing huge losses to the pig industry, especially in Guangdong with a high degree of industrialization. The prevention of PED mainly relies on the use of vaccines. However, since the emergence of the new PEDV strain in 2010, the commonly used PED inactivated and attenuated vaccines have not been very effective in immunization, and therefore, PED still exists in pig farms and recurs, which has caused serious harm to the healthy development of the pig industry. Existing studies have shown that the PEDV strain currently prevalent in China is a new genotype and is diversifying, and due to genetic variation, the existing vaccines are difficult to effectively prevent and control. At the same time, due to the difficulty of PEDV adapting to cells and low proliferation titer, the preparation of new and efficient vaccines is severely hindered, bringing great difficulties to the prevention of the disease.

[0005] Therefore, the cell culture medium conducive to PEDV proliferation not only helps to improve the culture titer of PEDV, but also facilitates the research and development of PEDV vaccine, and lays a foundation for industrialized cell culture production of vaccines, improvement of vaccine quality, and reduction of vaccine cost. SUMMARY

[0006] The present application provides a cell-based medium for proliferating porcine epidemic diarrhea virus, which is composed of the following components: glycine 30.0 mg / L, L-arginine hydrochloride 84.0 mg / L, L-cystine dihydrochloride 63.0 mg / L, L-glutamine 584.0 mg / L, L-histidine hydrochloride monohydrate 42.0 mg / L, L-isoleucine 105.0 mg / L, L-leucine 105.0 mg / L, L-lysine hydrochloride 146.0 mg / L, L-methionine 30.0 mg / L, L-phenylalanine 66.0 mg / L, L-serine 42.0 mg / L, L-threonine 95.0 mg / L, L-tryptophan 16.0 mg / L, L-tyrosine disodium salt dihydrate 104.0 mg / L, L-valine 94.0 mg / L, choline chloride 4.0 mg / L, dextrin calcium 4.0 mg / L, folic acid 4.0 mg / L, nicotinamide 4.0 mg / L, pyridoxine hydrochloride 4.0 mg / L, vitamin B2 0.4 mg / L, thiamine 4.0 mg / L, myo-inositol 7.2 mg / L, CaCl2 200.0 mg / L,

[0007]

[0008] Fe(NO3)3"9H2O 0.1mg / L, MgSO4 97.67mg / L, KC l 400mg / L, NaHCO3

[0009] 3700.0mg / L, NaC l 6400.0mg / L, NaH2PO4-H2O 125.0, D–glucose 4500.0mg / L, phenol red 15.0mg / L, sodium pyruvate 110.0mg / L.

[0010] ​The basic medium further comprises a "starvation medium" lacking glucose for experimental control, which is composed of glycine 30.0 mg / L, L-arginine hydrochloride 84.0 mg / L, L-cystine dihydrochloride 63.0 mg / L, L-glutamine 584.0 mg / L, L-histidine hydrochloride monohydrate 42.0 mg / L, L-isoleucine 105.0 mg / L, L-leucine 105.0 mg / L, L-lysine hydrochloride 146.0 mg / L, L-methionine 30.0 mg / L, L-phenylalanine 66.0 mg / L, L-serine 42.0 mg / L, L-threonine 95.0 mg / L, L-tryptophan 16.0 mg / L, L-tyrosine disodium salt dihydrate 104.0 mg / L, L-valine 94.0 mg / L, choline chloride 4.0 mg / L, calcium lactate 4.0 mg / L, folic acid 4.0 mg / L, nicotinamide 4.0 mg / L, pyridoxine hydrochloride 4.0 mg / L, vitamin B2 0.4 mg / L, thiamine 4.0 mg / L, myo-inositol 7.2 mg / L, CaCl2 200.0 mg / L, Fe(NO3)3 9H2O 0.1 mg / L, MgSO4 97.67 mg / L, KCl 400 mg / L, NaHCO3 3700.0 mg / L, NaCl 6400.0 mg / L, NaH2PO4-H2O 125.0.

[0011] Further, the L-glutamine concentration is 584 mg / L (corresponding concentration is 4 mM), and the glucose concentration is 4500 mg / L (corresponding concentration is 25 mM).

[0012] Further, the basic medium is one or several of MEM, DMEM, DMEM / F12.

[0013] Further, the final concentration of lactic acid is 5-30 mM.

[0014] Further, the medium further comprises a DMEM medium lacking glucose.

[0015] The application also provides a method for propagating porcine epidemic diarrhea virus using the above-mentioned medium.

[0016] Further, the cell inoculation density is 1×10 6 cells per well in a 12-well plate.

[0017] Further, the virus adsorption time is 2 h.

[0018] Further, the virus harvesting time is 24 h after inoculation.

[0019] The method has the advantages that the culture medium prepared by the method can greatly improve the proliferation speed of viruses and animal cells, and further improve the efficiency of related experiments. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure A is the relative fluorescence quantitative PCR method for determining the mRNA level of PEDV N gene, and Figure B is the protein electrophoretogram of the PEDV N protein level detected by western blot method; wherein, 1. PEDV GD / HZ / 2016 virus is proliferated in DMEM normal culture medium; 2. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose; 3. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose but with 20 mM of lactic acid; 4. Normal cell blank control group.

[0021] Figure 2 Figure A is the relative fluorescence quantitative PCR method for determining the mRNA level of PEDV N gene, and Figure B is the protein electrophoretogram of the PEDV N protein level detected by western blot method; wherein, 1. PEDV GD / HZ / 2016 virus is proliferated in DMEM normal culture medium; 2. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose but with 20 mM of lactic acid; 3. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose but with 20 mM of lactic acid; 4. Normal cell blank control group.

[0022] Figure 3 Figure A is the relative fluorescence quantitative PCR method for determining the mRNA level of PEDV N gene, and Figure B is the protein electrophoretogram of the PEDV N protein level detected by western blot method; wherein, 1. PEDV GD / HZ / 2016 virus is proliferated in DMEM normal culture medium; 2. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose but with 20 mM of lactic acid; 3. PEDV GD / HZ / 2016 virus is proliferated in DMEM culture medium without glucose but with 20 mM of lactic acid; 4. Normal cell blank control group.

[0023] Figure 4 Effect of lactic acid on proliferation of PEDV with different MOI (1. PEDV GD / HZ / 2016 virus proliferated in DMEM normal medium; 2. PEDV GD / HZ / 2016 virus proliferated in DMEM medium added with 20 mM lactic acid). DETAILED DESCRIPTION

[0024] Example 1, configuration of various culture media

[0025] Configuration of basic medium: 500 ml of DMEM containing glycine 30 mg / L, L-arginine hydrochloride 84 mg / L, L-cystine dihydrochloride 63 mg / L, L-glutamine 584 mg / L, L-histidine hydrochloride monohydrate 42 mg / L, L-isoleucine 105 mg / L, L-leucine 105 mg / L, L-lysine hydrochloride 146 mg / L, L-methionine 30 mg / L, L-phenylalanine 66 mg / L, L-serine 42 mg / L, L-threonine 95 mg / L, L-tryptophan 16 mg / L L-tyrosine disodium salt dihydrate 104 mg / L, L-valine 94 mg / L, choline chloride 4.0, calcium lactate 4 mg / L, folic acid 4 mg / L, nicotinamide 4 mg / L, pyridoxine hydrochloride 4 mg / L, vitamin B2 0.4 mg / L, thiamine 4 mg / L, myo-inositol 7.2 mg / L, CaCl2 200 mg / L, Fe(NO3)3·9H2O 0.1 mg / L, MgSO4 97.67 mg / L, KCl 400 mg / L, NaHCO3 3700 mg / L, NaCl 6400 mg / L, NaH2PO4-H2O 125 mg / L, D-glucose 4500 mg / L, phenol red 15 mg / L, sodium pyruvate 110 mg / L, 3 μM streptomycin- lincomycin mixture (Solarbio, P1400), 0.3% (volume ratio) tryptone phosphate broth, 0.02% (volume ratio) yeast extract.

[0026] Configuration of "starvation medium": 500 ml of DMEM containing glycine 30 mg / L, L-arginine hydrochloride 84 mg / L, L-cystine dihydrochloride 63 mg / L, L-glutamine 584 mg / L, L-histidine hydrochloride monohydrate 42 mg / L, L-isoleucine 105 mg / L, L-leucine 105 mg / L, L-lysine hydrochloride 146 mg / L, L-methionine 30 mg / L, L-phenylalanine 66 mg / L, L-serine 42 mg / L, L-threonine 95 mg / L, L-tryptophan 16 mg / L L-tyrosine disodium salt dihydrate 104 mg / L, L-valine 94 mg / L, choline chloride 4.0, calcium ractopamine 4 mg / L, folic acid 4 mg / L, nicotinamide 4 mg / L, pyridoxine hydrochloride 4 mg / L, vitamin B2 0.4 mg / L, thiamine 4 mg / L, myo-inositol 7.2 mg / L, CaCl2 200 mg / L, Fe(NO3)3"9H2O 0.1 mg / L, MgSO4 97.67 mg / L, KCl 400 mg / L, NaHCO3 3700 mg / L, NaCl 6400 mg / L, NaH2PO4-H2O 125 mg / L, sodium pyruvate 110 mg / L, 3 μM streptomycin mix (Solarbio, P1400), 0.3% (volume ratio) of tryptone broth, 0.02% (volume ratio) of yeast extract.

[0027] The base medium with 20 mM lactic acid was prepared as follows: 500 ml DMEM containing glycine 30 mg / L, L-arginine hydrochloride 84 mg / L, L-cystine dihydrochloride 63 mg / L, L-glutamine 584 mg / L, L-histidine hydrochloride monohydrate 42 mg / L, L-isoleucine 105 mg / L, L-leucine 105 mg / L, L-lysine hydrochloride 146 mg / L, L-methionine 30 mg / L, L-phenylalanine 66 mg / L, L-serine 42 mg / L, L-threonine 95 mg / L, L-tryptophan 16 mg / L, L-tyrosine disodium salt dihydrate 104 mg / L, L-valine 94 mg / L, choline chloride 4 mg / L, calcium pantothenate 4 mg / L, folic acid 4 mg / L, nicotinamide 4 mg / L, pyridoxine hydrochloride 4 mg / L, riboflavin 0.4 mg / L, thiamine 4 mg / L, myo-inositol 7.2 mg / L, CaCl2 200 mg / L, Fe(NO3)3"9H2O 0.1 mg / L, MgSO4 97.67 mg / L, KCl 400 mg / L, NaHCO3 3700 mg / L, NaCl 6400 mg / L, NaH2PO4-H2O 125 mg / L, phenol red 15 mg / L, sodium pyruvate 110 mg / L, 3 μM streptomycin mix (Solarbio, P1400), 0.3% (v / v) tryptone broth, 0.02% (v / v) yeast extract, final concentration of lactic acid 20 mM.The configuration of the improved culture medium: 500ml of DMEM contains glycine 30mg / L, L-arginine hydrochloride 84mg / L, L-cystine dihydrochloride 63mg / L, L-glutamine 584mg / L, L-histidine hydrochloride monohydrate 42mg / L, L-isoleucine 105mg / L, L-leucine 105mg / L, L-lysine hydrochloride 146mg / L, L-methionine 30mg / L, L-phenylalanine 66mg / L, L-serine 42mg / L, L-threonine 95mg / L, L-tryptophan 16mg / L L-tyrosine disodium salt dihydrate 104mg / L, L-valine 94mg / L, choline chloride 4.0, calcium lactate 4mg / L, folic acid 4mg / L, nicotinamide 4mg / L, pyridoxine hydrochloride 4mg / L, vitamin B2 0.4mg / L, thiamine 4mg / L, myo-inositol 7.2mg / L, CaCl2 200mg / L, Fe(NO3)3"9H2O 0.1mg / L, MgSO4 97.67mg / L, KCl 400mg / L, NaHCO3 3700mg / L, NaCl 6400mg / L, NaH2PO4-H2O 125mg / L, D-glucose 4500mg / L, phenol red 15mg / L, sodium pyruvate 110mg / L, 3μM streptomycin mixture (Solarbio, P1400), 0.3% (volume ratio) trypsin phosphate broth, 0.02% (volume ratio) yeast extract, and the final concentration of lactic acid is 5-30mM.

[0028] Example 2, lactic acid can promote the proliferation of PEDV virus verification experiment

[0029] After determining the cell seeding density (1×10 6 Lactic acid is one of the key intermediates of the tricarboxylic acid cycle, and the appropriate addition of lactic acid is often beneficial to the proliferation of viruses. After determining the parameters, lactic acid was added at different concentrations (2, 5, 10, 20, 30mM) to study and analyze the promoting effect of lactic acid on PEDV GD / HZ / 2016 strain.

[0030] First, the CCK-8 assay was used to examine the toxicity of lactate to Vero cells. The results showed that lactate at a concentration as high as 23.06 mM had no toxic effect on the cells. Furthermore, based on the metabolic pathway, it was found that one glucose molecule is broken down into two lactate molecules through glycolysis. "Starvation medium" was supplemented with 20 mM lactate, and then the proliferation of PEDV in the following three media (basal medium, "starvation medium," and "starvation medium" supplemented with 20 mM lactate) was analyzed. The mRNA and protein levels of the PEDV N gene were analyzed using relative quantitative PCR and Western blotting methods, respectively. The experimental methods are as follows:

[0031] (1) Relative quantitative PCR: Cell samples that proliferated PEDV in the above three culture media were collected respectively. Total RNA samples of each group were extracted using the MiniBEST Universal RNA Extraction Kit (catalog number 9767) from Takara. Relative quantitative PCR was used for detection and analysis using the HiScript II One Step qRT-PCR SYBR Green Kit (catalog number: Q221-01) from Nanjing Novizan Biotechnology Co., Ltd.

[0032] (2) Western blot method: Protein samples of PEDV cells proliferated in the above three culture media were collected. Cells were lysed with RI PA lysis buffer containing PMSF, centrifuged, and the supernatant was collected. The protein concentration of each group of samples was determined by BCA method, and the expression of PEDV N protein was detected by Western blot. First, the protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes by electroporation. The membranes were blocked with 5% skim milk powder at 37°C for 1 hour. Then, the membranes were incubated with mouse N monoclonal antibody (1:1000) and HRP-labeled goat anti-mouse IgG (dilution ratio 1:5000) was used as the secondary antibody. The results were observed by ECL imaging and photographed.

[0033] The results showed that, as expected, the PEDV titer in the lactate-supplemented "starved medium" group recovered and exceeded that of the basal medium group compared to the "starved medium" group. Figure 1 To determine the lactate concentration that produced the most significant promoting effect, we added lactate to the starved medium in a gradient manner. The results are as follows: Figure 2 As shown. The results indicate that adding 20 mM lactate to Vero cells infected with PEDV GD / HZ / 2016 produced the greatest promoting effect ( Figure 2 ).

[0034] Example 3: Lactic acid can promote the proliferation of different genotypes of PEDV virus.

[0035] To demonstrate the ability of lactic acid to promote PEDV of other strains, PEDV GD / HZ / 2016 and PEDV TB strains belonging to GI II genotype isolated from clinical samples, and CV777 strain belonging to GI genotype were experimented respectively. The above 3 strains of viruses were proliferated in basal medium and modified medium respectively, and the TCI D 50 Method determination as follows: each group of cell suspension samples were collected, and then TCI D 50 determination was carried out on Vero cells. Each group of virus suspension samples diluted by 10 times successively (10 -1 to 10 -9 ) were inoculated in 96-well with monolayer Vero cells, and after 4-5 days of culture in 5% CO2 incubator at 37°C, the cytopathic effect of cells was checked. The virus titer of each group of sample virus liquid was calculated according to Reed-Muench equation, and analyzed. The results are shown in Figure 3 , the virus was collected 72 hours after infection, and the virus mRNA and titer determination showed that lactic acid could promote the proliferation of PEDV regardless of genotype Figure 3 A-C).

[0036] Example 4, lactic acid promotes the replication of PEDV in Vero cells is not affected by MOI

[0037] To confirm that lactic acid has the same effect on promoting PEDV GD / HZ / 2016 strain with different MOI, we inoculated PEDV GD / HZ / 2016 infected Vero cells at MOI of 0.01, 0.001 and 0.0001. The above 3 different MOI of PEDV were proliferated in basal medium and modified medium respectively, and the TCI D 50 method in Example 3, the PEDV virus titer level was analyzed. The results are shown in Figure 4 , the results show that lactic acid has a promoting effect on PEDV GD / HZ / 2016 strain with different MOI Figure 4 A-C).

Claims

1. A cell culture medium for propagating porcine epidemic diarrhea virus, the cell culture medium being made of the following ingredients: glycine 30 mg / L, L-arginine hydrochloride 84 mg / L, L-cystine dihydrochloride 63 mg / L, L-glutamine 584 mg / L, L-histidine hydrochloride monohydrate 42 mg / L, L-isoleucine 105 mg / L, L-leucine 105 mg / L, L-lysine hydrochloride 146 mg / L, L-methionine 30 mg / L, L-phenylalanine 66 mg / L, L-serine 42 mg / L, L-threonine 95 mg / L, L-tryptophan 16 mg / L, L-tyrosine disodium salt dihydrate 104 mg / L, L-valine 94 mg / L, choline chloride 4 mg / L, calcium pantothenate 4 mg / L, folic acid 4 mg / L, nicotinamide 4 mg / L, pyridoxine hydrochloride 4 mg / L, vitamin B2 0.4 mg / L, thiamine 4 mg / L, myo-inositol 7.2 mg / L, CaCl2 200 mg / L, Fe(NO3)3·9H2O 0.1 mg / L, MgSO4 97.67 mg / L, KCl 400 mg / L, NaHCO3 3700 mg / L, NaCl 6400 mg / L, NaH2PO4·H2O 125 mg / L, phenol red 15 mg / L, sodium pyruvate 110 mg / L, 3 μM streptomycin mix, 0.3% trypticase phosphate broth, 0.02% yeast extract, and a final concentration of 20 mM lactic acid; and the cell is a Vero cell.

2. A cell culture method for promoting the propagation of porcine epidemic diarrhea virus, characterized by, The porcine epidemic diarrhea virus is cultured in the cell culture medium of claim 1, and the cell is a Vero cell.

3. The cell culture method of claim 2, wherein, Cell seeding density was 1 x 6 10 cells / well in 12-well plates.

4. The cell culture method of claim 2, wherein, The virus adsorption time is 2 h.

5. The cell culture method of claim 2, wherein, The virus harvesting time is 24 h after inoculation. The virus adsorption time is 2 h. The virus harvesting time is 24 h after inoculation.

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