A kind of influenza virus maintenance liquid formula and its preparation method and application

Abstract

The present invention discloses a formula of influenza virus maintenance solution and its preparation method and application, and specifically relates to the field of biomedicine technology. The virus maintenance solution includes DMEM / F-12 basal culture medium and active ingredients, and the active ingredients include amino acids, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter includes yeast extract. The present invention develops an influenza virus maintenance solution formula based on DMEM / F-12 basal culture medium by establishing an influenza virus subunit ELISA detection kit and taking the ELISA test result at the culture end point as the response value. The virus yield of influenza virus on MDCK-v cell matrix can be significantly improved, and production costs can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a formula of an influenza virus maintenance solution, a preparation method thereof, and an application thereof. Background Art

[0002] Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12) is a widely used basal culture medium supplemented with serum to support the growth of many different mammalian cell types. Cells that have been successfully cultured in DMEM / F-12 include MDCK, glial cells, fibroblasts, human endothelial cells, and rat fibroblasts.

[0003] DMEM / F-12 is a 1:1 mixture of DMEM and Ham's F-12. This formula contains the high concentration of glucose, amino acids, and vitamins found in DMEM along with various components of F-12. However, DMEM / F-12 does not contain proteins, lipids, or growth factors.

[0004] Human influenza is a disease caused by influenza viruses infecting humans, resulting in millions of severe cases and hundreds of thousands of deaths each year. It poses a significant threat to human health and has become a major public health issue of global concern. Currently, the main human influenza vaccines used internationally include subunit vaccines based on purified live, inactivated, or recombinant viruses. Due to the strong mutation capacity of influenza viruses and the blocking effect of human antibodies, differences exist between existing vaccine strains and prevalent strains, reducing the vaccine's immune efficacy and protection rate. Whole-genome sequencing is currently commonly used to determine the antigenicity of newly isolated influenza viruses, and developing new vaccines based on this requires a long period of time.

[0005] The fluid used for virus culture is typically called viral culture medium or viral maintenance medium. Its composition and preparation method directly determine cell survival and whether sufficient numbers of viral particles capable of infection and replication can be obtained. Viral maintenance medium typically uses serum-free basal media such as DMEM, MEM, DMEM / F-12, M199, and 1640. These basal media vary in their effectiveness in culturing influenza viruses, leaving much room for improvement. Summary of the Invention

[0006] To this end, the present invention provides an influenza virus maintenance liquid formula and a preparation method and application thereof to solve the above-mentioned problems.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] According to the first aspect of the present invention, an influenza virus maintenance solution is provided, comprising a DMEM / F-12 basal culture medium and active ingredients, wherein the active ingredients include amino acid sources, vitamins, salts, virus proliferation promoters, and additives, wherein the virus proliferation promoter comprises a yeast induction culture extract.

[0009] In the present invention, the osmotic pressure in the influenza virus maintenance solution is 320 to 330 mOsm / kg.

[0010] Furthermore, the amino acid source includes L-aspartic acid monohydrate, L-glutamine, L-arginine hydrochloride, L-cystine hydrochloride, L-lysine hydrochloride, L-proline and L-serine. As an example, preferably, L-aspartic acid monohydrate 1-3 mg / L, L-glutamine 50-80 mg / L, L-arginine hydrochloride 10-40 mg / L, L-cystine hydrochloride 4-8 mg / L, L-lysine hydrochloride 16-20 mg / L, L-proline 8-12 mg / L and L-serine 4-8 mg / L; more preferably, L-aspartic acid monohydrate 2 mg / L, L-glutamine 70 mg / L, L-arginine hydrochloride 20 mg / L, L-cystine hydrochloride 6 mg / L, L-lysine hydrochloride 18 mg / L, L-proline 10 mg / L, and L-serine 6 mg / L.

[0011] Furthermore, the vitamins include biotin, D-calcium pantothenate, nicotinamide, and inositol. As an example, preferably, biotin 0.0002-0.0006 mg / L, D-calcium pantothenate 0.2-0.6 mg / L, nicotinamide 0.2-0.6 mg / L, and inositol 2-30.2-0.6 mg / L; more preferably, biotin 0.0004 mg / L, D-calcium pantothenate 0.4 mg / L, nicotinamide 0.4 mg / L, and inositol 2.5 mg / L.

[0012] Furthermore, the additive includes D(+)-galactose. As an example, D(+)-galactose is preferably 100-300 mg / L, and more preferably 200 mg / L.

[0013] Furthermore, the salts include anhydrous calcium chloride and sodium chloride. As an example, 5-15 mg / L of anhydrous calcium chloride and 200-300 mg / L of sodium chloride are preferred; more preferably, 10 mg / L of anhydrous calcium chloride and 250 mg / L of sodium chloride are preferred.

[0014] Furthermore, the yeast induction culture extract is prepared by adding L-phenylalanine and citric acid to YPD liquid culture medium and then inoculating brewer's yeast, culturing for the first time for 12-24 hours, then adding glycerol, fennel extract and betaine to continue induction culture for 12-24 hours, and extracting yeast extract after the induction culture is completed.

[0015] As an example, the amount of L-phenylalanine added is 0.2-0.6 g / L, the amount of fennel extract added is 0.5-2 g / L, glycerol is 20-30 g / L, citric acid is 20-40 mM, and betaine is 0.04-0.08 g / L; more preferably, the amount of L-phenylalanine added is 0.4 g / L, the amount of fennel extract added is 1 g / L, glycerol is 25 g / L, citric acid is 25 mM, and betaine is 0.06 g / L.

[0016] Furthermore, the yeast extract extraction method includes adding sodium chloride and protease after the culture is completed; adjusting the pH, adding polysaccharidase after autolysis, and continuing autolysis; inactivating the enzyme, and centrifuging to obtain a yeast extract; vacuum concentrating the yeast extract and spray drying it to obtain a powdered yeast extract.

[0017] As an example, a 2-5% weight / volume suspension is prepared; 1-3% weight / volume sodium chloride and a protease are added; the pH is adjusted to 7-8, and after autolysis at 40-50°C for 12-24 hours, a polysaccharidase is added and the autolysis is continued for 12-24 hours; the enzyme is inactivated, and the yeast extract is obtained by centrifugation; the yeast extract is vacuum concentrated and then spray-dried to obtain a powdered yeast extract; the protease is papain, used in an amount of 500 U / mL; and the polysaccharidase is amylase, used in an amount of 500 U / mL.

[0018] As an example, the preferred amount of yeast induction culture extract added is 200-400 mg / L, and more preferably the amount of yeast induction culture extract added is 300 mg / L.

[0019] According to a second aspect of the present invention, a method for preparing the influenza virus maintenance solution as described above is provided, comprising: mixing DMEM / F-12 basal culture medium and active ingredients to obtain the solution, wherein the active ingredients include amino acid sources, vitamins, salts, virus proliferation promoters, and additives, wherein the virus proliferation promoter includes a yeast induction culture extract.

[0020] The other ingredients are the same as those of influenza virus maintenance fluid and will not be described again.

[0021] According to the third aspect of the present invention, an influenza virus maintenance liquid is provided for use in preparing influenza virus vaccines. As an example, it is mainly used for influenza B virus Bv, influenza B virus By, influenza A virus H1N1 and influenza A virus H3N2.

[0022] The present invention has the following advantages:

[0023] 1. The present invention adds yeast induction culture extract and screens the raw material ratio of the influenza virus maintenance solution to obtain an influenza virus maintenance solution that can promote the intracellular amplification of influenza virus, significantly increase the virus yield, and maintain a high virus titer, significantly improving the efficiency of the production process and reducing production costs;

[0024] 2. The preparation method of the present invention is simple, the harvested virus liquid has a high titer, and the batches are stable. It greatly reduces the use of manual labor, has simple operation steps, and has a low risk of contamination. It can reduce the floor space and space occupied by the production workshop, further reducing the production cost of the enterprise. It has low cost and high efficiency, and the safety of the obtained product is guaranteed, which is conducive to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0026] The structures, proportions, sizes, etc. illustrated in this specification are intended only to complement the contents disclosed herein and to facilitate understanding and reading by persons familiar with the art. They are not intended to limit the conditions under which the present invention may be implemented and therefore have no substantive technical significance. Any structural modifications, changes in proportions, or adjustments in sizes, without affecting the efficacy and objectives of the present invention, shall still fall within the scope of the technical contents disclosed herein.

[0027] Figure 1 A comparison chart of tryptophan and glutcystine content in yeast induced culture and non-induced culture provided by the present invention; wherein A is the tryptophan content in g / L; B is the glutathione content in mg / L;

[0028] Figure 2 Interactive analysis diagram for screening raw materials for influenza virus maintenance solution provided in Example 1 of the present invention;

[0029] Figure 3 The mouse Bv peak graph provided in Experimental Example 1 of the present invention;

[0030] Figure 4 The mouse By peak graph provided in Experimental Example 1 of the present invention;

[0031] Figure 5 The mouse H1N1 peak graph provided in Experimental Example 1 of the present invention;

[0032] Figure 6 The mouse H3N2 peak diagram provided in Experimental Example 1 of the present invention;

[0033] Figure 7 Rabbit Bv peak graph provided for Experimental Example 1 of the present invention;

[0034] Figure 8 Rabbit By peak diagram provided in Experimental Example 1 of the present invention;

[0035] Figure 9 Rabbit H1N1 peak graph provided in Experimental Example 1 of the present invention;

[0036] Figure 10 Rabbit H3N2 peak diagram provided for Experimental Example 1 of the present invention;

[0037] Figure 11 This is a linear relationship diagram of Bv provided in Experimental Example 1 of the present invention;

[0038] Figure 12 This is the By-related linear relationship diagram provided in Experimental Example 1 of the present invention;

[0039] Figure 13 This is the H1N1 related linear relationship diagram provided in Experimental Example 1 of the present invention;

[0040] Figure 14 This is a linear relationship diagram of H3N2 provided in Experimental Example 1 of the present invention;

[0041] Figure 15 The dilution factors provided for Experimental Example 1 of the present invention are shown in the layout diagram and the plating diagram;

[0042] Figure 16 A comparison chart of the hemagglutination titers of influenza virus cultured in influenza virus maintenance medium and DMEM / F-12 basal medium provided in Experimental Example 3 of the present invention;

[0043] Figure 17 This is a comparison chart of the hemagglutination titers of influenza virus cultured in influenza virus maintenance medium and DMEM / F-12 basal medium provided in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0044] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0045] Except for the raw materials used in the present invention that are individually marked, the other raw materials are purchased from Merck Biotechnology and McLean's corresponding products with CAS numbers, which are not listed one by one.

[0046] Preparation method of yeast induced culture extract:

[0047] YPD liquid medium (HB5193-1) was added with 0.4 g / L L-phenylalanine (CAS: 2577-40-4) and 25 mM citric acid, and then inoculated with brewer's yeast (HZB119485). Other culture conditions were the same as those for normal brewer's yeast culture conditions. The first culture was 12-24 hours, and then glycerol, fennel extract (CAS: 84775-42-8) and betaine were added for further induction culture for 12-24 hours. The culture was continued, and after the end of the culture, a 2-5% weight / volume suspension was prepared; 1-3% weight / volume sodium chloride and a protease were added; the pH was adjusted to 7-8, and after autolysis at 40-50° C. for 12-24 hours, a polysaccharidase was added, and the autolysis was continued for 12-24 hours; the enzyme was inactivated, and the yeast extract was obtained by centrifugation; the yeast extract was vacuum concentrated and spray-dried to obtain a powdered yeast extract; the protease was papain at a dosage of 500 U / mL; and the polysaccharidase was amylase at a dosage of 500 U / mL.

[0048] Control group: normal culture without adding inducers to measure the tryptophan and glutcystine contents.

[0049] The tryptophan and glutcystine contents after induced culture and non-induced culture are as follows Figure 1 As shown by Figure 1 It can be seen that the content of tryptophan and glutcystine increased significantly after induction culture.

[0050] TPCK pancreatic enzyme solution 1g / L: Dissolve TPCK pancreatic enzyme (CAS#: LS003741; LOT#: 33P24072) in 1mM hydrochloric acid, sterile filter, and store at 2-8°C for 6 months. Add TPCK pancreatic enzyme solution before subsequent virus culture at a working concentration of 2μg / ml culture volume.

[0051] Example 1

[0052] This embodiment provides an influenza virus maintenance solution, and the screening process of raw material types:

[0053] Type A: IVR-215 (C1); Type B: B / Phuket / 3073 / 2013 (C1).

[0054] We investigated the effects of optimizing the viral maintenance solution formulation on viral yield. The model used was a Two-Level 8-4IV factorial analysis. The components investigated are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058]

[0059] The 49 components in Table 1 were preliminarily screened; the concentrations of the preliminarily screened components were optimized; and the final determined components were interactively analyzed. Figure 2 .

[0060] The indicators of the model are shown in Table 2.

[0061] Table 2

[0062] SS DF MS F-number P-value Model 16.21 8 2.03 7.94 ＜0.0001 AA 0.0707 1 0.0707 0.2770 0.6037 CC 0.0585 1 0.0585 0.2293 0.6366 EE 1.11 1 1.11 4.35 0.0483 KK 3.67 1 3.67 14.39 0.0009 LL 2.32 1 2.32 9.09 0.0062 OO 3.09 1 3.09 12.10 0.0020 PP 4.13 1 4.13 16.20 0.0005 AC 1.76 1 1.76 6.88 0.0152 Remnants 5.87 23 0.2552 Total deviation 22.08 31

[0063] The optimal combination of influenza virus maintenance fluid was finally determined to be: DMEM / F-12 supplemented with L-aspartic acid monohydrate, L-glutamine, L-arginine hydrochloride, L-cystine hydrochloride, L-lysine hydrochloride, L-proline, L-serine, biotin, D-pantothenate calcium, nicotinamide, inositol, D(+)-galactose, yeast induction culture extract, anhydrous calcium chloride and sodium chloride.

[0064] Example 2

[0065] This embodiment provides an influenza virus maintenance solution:

[0066] Active ingredients: L-aspartic acid monohydrate 2mg / L, L-glutamine 70mg / L, L-arginine hydrochloride 20mg / L, L-cystine hydrochloride 6mg / L, L-lysine hydrochloride 18mg / L, D(+)-galactose 200mg / L, L-proline 10mg / L, L-serine 6mg / L, niacinamide 0.4mg / L, anhydrous calcium chloride 10mg / L, inositol 2.5mg / L, D-calcium pantothenate 0.4mg / L, biotin 0.0004mg / L, yeast induced culture extract 300mg / L, sodium chloride 250mg / L.

[0067] DMEM / F-12 basal culture medium.

[0068] The DMEM / F-12 basal culture medium and the active ingredients are mixed and then disinfected to obtain the influenza virus maintenance solution.

[0069] Example 3

[0070] This embodiment provides an influenza virus maintenance solution:

[0071] Active ingredients: L-aspartic acid monohydrate 1mg / L, L-glutamine 50mg / L, L-arginine hydrochloride 12mg / L, L-cystine hydrochloride 4mg / L, L-lysine hydrochloride 16mg / L, D(+)-galactose 100mg / L, L-proline 8mg / L, L-serine 4mg / L, nicotinamide 0.2mg / L, anhydrous calcium chloride 5mg / L, inositol 2mg / L, D-calcium pantothenate 0.2mg / L, biotin 0.0002mg / L, yeast induced culture extract 120mg / L, sodium chloride 200mg / L.

[0072] DMEM / F-12 basal culture medium.

[0073] The DMEM / F-12 basal culture medium and the active ingredients are mixed and then disinfected to obtain the influenza virus maintenance solution.

[0074] Example 4

[0075] This embodiment provides an influenza virus maintenance solution:

[0076] Active ingredients: L-aspartic acid monohydrate 3mg / L, L-glutamine 80mg / L, L-arginine hydrochloride 40mg / L, L-cystine hydrochloride 8mg / L, L-lysine hydrochloride 20mg / L, D(+)-galactose 300mg / L, L-proline 12mg / L, L-serine 8mg / L, niacinamide 0.6mg / L, anhydrous CaCl2 15mg / L, inositol 3mg / L, D-calcium pantothenate 0.6mg / L, biotin 0.0006mg / L, yeast induced culture extract 400mg / L, sodium chloride 300mg / L.

[0077] DMEM / F-12 basal culture medium.

[0078] The DMEM / F-12 basal culture medium and the active ingredients are mixed and then disinfected to obtain the influenza virus maintenance solution.

[0079] Experimental Example 1

[0080] Influenza B virus Bv, influenza B virus By, influenza A virus H1N1, and influenza A virus H3N2 (all viruses were purchased from NIBSC, the same applies hereinafter) were cultured using the influenza virus maintenance medium of Example 2;

[0081] Develop ELISA quantitative detection kits for IVR-215 (H1N1), SAN-010 (H3N2), BVR-26 (Bv), and B / Phuket / 3073 / 2013 (By) strains;

[0082] Five mice (MF59 adjuvant) and one rabbit (Freund's adjuvant) were immunized with subunit antigens of each viral strain. Rabbit serum was used as the capture antibody, and mouse serum as the detection antibody. An ELISA detection system was established.

[0083] Rabbit serum and mouse serum: According to the standards, rabbit immune serum and mouse immune serum were extracted and purified respectively and set aside.

[0084] Specific: 1. Prepare female New Zealand rabbits weighing 2-3 kg, 4-5 months old.

[0085] 2. Use Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for the second and third immunizations. Allow 2 weeks between immunizations. Inject subcutaneously at multiple points in the back and groin of the rabbit, 1 ml per rabbit, typically at 5-7 subcutaneous sites.

[0086] 3. Record a free time.

[0087] 4. Record the second free time.

[0088] 5. Record the three free times.

[0089] 6. One week after the third vaccination, collect whole blood from the carotid artery or heart of each of the four rabbits. Record the time of blood collection.

[0090] 7. After blood collection, separate the serum by placing the blood at 37°C for 30-60 minutes to allow it to coagulate. The clot should separate from the surface of the collector and then be placed at 4°C overnight to allow the clot to shrink. Keep the clot suspended as much as possible overnight, and collect the serum by gravity. The next day, centrifuge at 3000 rpm for 10 minutes, remove the serum, and incubate in a 56°C water bath for 30 minutes.

[0091] 8. After the serum was filtered through a 0.45 μm filter membrane, IgG antibodies were purified using a 5 ml Protein G affinity chromatography column.

[0092] 9. Purify mouse serum and rabbit serum according to the purification steps, and record the volume information, time information, and solution preparation information.

[0093] Mouse serum purification:

[0094] 10. Prepare the following solution required for purification:

[0095] (1) 10X binding / washing buffer: 2 mol / L sodium phosphate buffer, pH 7.4; [1.5 M NaCl, 200 mM Na2HPO4, pH 7.4] (2) Binding / washing buffer: 200 mmol / L sodium phosphate buffer, pH 7.4; [0.15 M NaCl, 20 mM Na2HPO4, pH 7.4] (3) Elution buffer: 100 mmol / L citric acid buffer, pH 2.6; [0.1 M citric acid, pH 2.5-3.0] (4) Neutralization buffer: 1 mol / L Tris-HCl solution; (5) Dialysis buffer: 10 mmol / L PBS, pH 7.4.

[0096] 11. Dialysis bag handling: (1) Cut the dialysis bag into small pieces of appropriate length (10-20 cm). (2) Boil the dialysis bag in a large volume of 2% (W / V) sodium bicarbonate and 1mmol / L EDTA (pH 8.0) for 10 minutes. (3) Rinse the dialysis bag thoroughly with distilled water. (4) Boil it in 1mmol / L EDTA (pH 8.0) for 10 minutes. (5) After cooling, store it at 4 degrees, making sure that the dialysis bag is always immersed in the solution. From this point on, gloves must be worn when handling the dialysis bag. (6) Fill the dialysis bag with water before use and then drain it to clean it.

[0097] 12. AKTA protein purification system operation steps: (1) It is recommended to filter the water and buffer using a 0.22μm or 0.45μm filter membrane before use. (2) Sample preparation: Before loading the column, ensure that the sample solution has the appropriate ionic strength and pH value. The serum can be diluted 1:1 with binding / wash buffer or dialyzed overnight in binding / wash buffer. (3) Add 10X binding buffer to the serum to be purified at a serum:buffer volume ratio of 9:1, adjust the serum pH to 7.4, and aspirate into a 5mL sterile syringe. (4) Remove the Protein G affinity chromatography column from the refrigerator, equilibrate to room temperature, and connect it to the purification system. (5) Cleaning of purification pipelines and chromatography columns: Place tubes A and B in injection water, flush the pipelines to empty the preservation solution, and continue to flush for 5 times the column volume. Place tube A in binding buffer and tube B in elution buffer. Set the parameter to "Inlet A" and the system flow rate to 15 mL / min. Add about 3 times the column volume of buffer to flush the chromatography column and observe whether the pressure is abnormal or whether bubbles are generated; change the column position to "by pass" and flush the peripheral pipeline with the same flow rate of about 3 times the column volume of buffer. (6) Set the parameter to "Inlet B" and the system flow rate to 5 mL / min. Flush the chromatography column and pipeline with 100% B elution buffer, and let about 3 times the column volume of elution buffer flow through. (7) Equilibration of chromatography column: Switch the parameter back to "Inlet A" and the system flow rate to 5 mL / min. Allow the binding buffer to equilibrate the chromatography column. Observe that the pH is stable at 7.4 and the UV absorbance baseline remains stable, then switch back to "by pass" and equilibrate the peripheral pipeline. (8) Set the purification program: the sample volume is 5 mL, the binding solution volume is 20 mL, the elution solution volume is 10 mL, the system flow rate is 5 mL / min, and the program is started. (9) Observe the peak graph. When the pH value begins to decrease and the UV absorbance value increases rapidly, use a clean 15 mL centrifuge tube to collect the sample at the elution peak. Discard the sample before and after the peak appropriately. Generally, use UV80 as the boundary and collect the peak greater than UV80. (10) Rinse the chromatography column and tubing with 5 column volumes of binding buffer and balance the pH to 7.4. If the sample is loaded again, repeat steps ④ to ⑧. (11) In-place cleaning: Use 3 column volumes of binding / washing buffer, 5 column volumes of deionized water, and 5 column volumes of 20% ethanol to wash the resin in sequence. Remove the chromatography column and store it at 2-8°C, away from the frost side to avoid freezing. (12) Attach the peak graph, save the electronic file, and print the paper file. Turn off the purification system. (13) Immediately add neutralization buffer to the collected sample to adjust the pH to 7.4 (monitor with pH test paper) and mix thoroughly. (14) Transfer the antibody to a dialysis bag and dialyze overnight in 2L PBS buffer at 4°C, changing the dialysate every 4 hours for three times.

[0098] Rabbit serum purification:

[0099] 13. Prepare the following solutions for purification: (1) 10X binding / washing buffer: 2 mol / L sodium phosphate buffer, pH 7.4; [1.5 M NaCl, 200 mM Na2HPO4, pH 7.4] (2) Binding / washing buffer: 200 mmol / L sodium phosphate buffer, pH 7.4; [0.15 M NaCl, 20 mM Na2HPO4, pH 7.4] (3) Elution buffer: 100 mmol / L citric acid buffer, pH 2.6; [0.1 M citric acid, pH 2.5-3.0] (4) Neutralization buffer: 1 mol / L Tris-HCl solution; (5) Dialysis buffer: 10 mmol / L PBS, pH 7.4.

[0100] 14. Dialysis bag handling: (1) Cut the dialysis bag into small pieces of appropriate length (10-20 cm). (2) Boil the dialysis bag in a large volume of 2% (W / V) sodium bicarbonate and 1mmol / L EDTA (pH 8.0) for 10 minutes. (3) Rinse the dialysis bag thoroughly with distilled water. (4) Boil it in 1mmol / L EDTA (pH 8.0) for 10 minutes. (5) After cooling, store it at 4 degrees, making sure that the dialysis bag is always immersed in the solution. From this point on, gloves must be worn when handling the dialysis bag. (6) Fill the dialysis bag with water before use and then drain it to clean it.

[0101] 15. AKTA protein purification system operation steps: (1) It is recommended to filter the water and buffer using a 0.22μm or 0.45μm filter membrane before use. (2) Sample preparation: Before loading the column, ensure that the sample solution has the appropriate ionic strength and pH value. The serum can be diluted 1:1 with binding / wash buffer or dialyzed overnight in binding / wash buffer. (3) Add 10X binding buffer to the serum to be purified at a serum:buffer volume ratio of 9:1, adjust the serum pH to 7.4, and aspirate into a 5mL sterile syringe. (4) Remove the Protein G affinity chromatography column from the refrigerator, equilibrate to room temperature, and connect it to the purification system. (5) Cleaning of purification pipelines and chromatography columns: Place tubes A and B in injection water, flush the pipelines to empty the preservation solution, and continue to flush for 5 times the column volume. Place tube A in binding buffer and tube B in elution buffer. Set the parameter to "Inlet A" and the system flow rate to 15 mL / min. Add about 3 times the column volume of buffer to flush the chromatography column and observe whether the pressure is abnormal or whether bubbles are generated; change the column position to "by pass" and flush the peripheral pipeline with the same flow rate of about 3 times the column volume of buffer. (6) Set the parameter to "Inlet B" and the system flow rate to 5 mL / min. Flush the chromatography column and pipeline with 100% B elution buffer, and let about 3 times the column volume of elution buffer flow. (7) Equilibration of chromatography column: Switch the parameter back to "Inlet A" and the system flow rate to 5 mL / min. Allow the binding buffer to equilibrate the chromatography column. Observe that the pH is stable at 7.4 and the UV absorbance baseline remains stable, then switch back to "by pass" and equilibrate the peripheral pipeline. (8) Set up the purification program: the sample volume is 5 mL, the binding liquid volume is 20 mL, the elution liquid volume is 10 mL, the system flow rate is 5 mL / min, and the program is started. (9) Observe the result peak graph. When the pH value begins to decrease and the UV absorbance value increases rapidly, use a clean 15 mL centrifuge tube to collect the sample at the elution peak. Discard the samples before and after the peak appropriately. Generally, use UV80 as the boundary and collect the peak greater than UV80. (10) Rinse the chromatography column and pipeline with 5 times the column volume of binding buffer and balance the pH to 7.4. If the sample is loaded again, repeat steps ④ to ⑧. (11) In-situ cleaning: Use 3 times the column volume of binding / washing buffer, 5 times the column volume of deionized water, and 5 times the column volume of 20% ethanol to wash the resin in sequence. Remove the chromatography column and store it at 2-8°C, away from the frost side to avoid freezing. (12) Attached peak graph as shown Figure 3-10 , save the electronic file and print the paper file. Turn off the purification system. (13) Immediately add neutralization buffer to the collected sample to adjust the pH to 7.4 [monitor with pH test paper] and mix thoroughly. (14) Transfer the antibody to a dialysis bag and dialyze overnight in 2L PBS buffer at 4°C, changing the dialysate every 4 hours for 3 times.

[0102] 16. Take samples for protein electrophoresis purity analysis and protein content determination. Aliquot the remaining sample into two aliquots for freezing. Aliquot the rabbit serum into 1 ml and 0.1 ml vials. Aliquot the mouse serum into two aliquots for HRP labeling, for a total of four aliquots. Record the volume and aliquot date.

[0103] HRP labeling

[0104] 17. Label an aliquot of mouse serum with HRP.

[0105] 18. Prepare carbonate buffer (CBS) (0.05 M): Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, dissolve them in WFI to make up to 1 L, and adjust the pH to 9.6 with 1 M NaOH.

[0106] 19. Dilute the antibody to 2 mg / ml with CB (pH 9.6) (BCA can be used for calibration), and take 1 ml of each solution into a dialysis bag; dialyze with CB at 4°C, changing the solution every 2 hours (3 times is required) and set aside.

[0107] 20. Enzyme activation (keep away from light throughout the process): (1) Weigh 30 mg of HRP (in a 4 ml EP tube) and dissolve it in 1.5 ml of ultrapure water (concentration: 20 mg / ml). (2) Weigh 30 mg of NaIO4 (in a 4 ml EP tube) and dissolve it in 1.2 ml of ultrapure water (concentration: 25 mg / ml). (3) Take 1 ml of the above HRP solution in a 4 ml EP tube, and then take 800 μL of NaIO4 solution and add it dropwise while shaking. Place the mixed solution at 4°C in the dark for 30 minutes. (4) Take 200 μL of ethylene glycol and add it dropwise to the above solution while shaking. Place it at 4°C in the dark for 30 minutes. (Terminate activation). (The volume ratio of HRP: NaIO4: ethylene glycol is 5:4:1)

[0108] 21. Antibody-HRP Conjugation: Add 200 μL of activated HRP to each dialysis bag containing the monoclonal antibody. Dialyze against CB, changing the solution every 2 hours (three times). Incubate overnight at 4°C in the dark. Collect the solution in each dialysis bag using a 4 mL EP tube. (Mass ratio of Ab:HRP:NaIO4 is 1:1:1).

[0109] 22. Stop the reaction: Prepare NaBH4: Add 2ml of ultrapure water to a 4ml EP tube. Weigh 4mg of NaBH4 and dissolve it in the tube (protect from light). Add 80μL of the NaBH4 solution to each EP tube from step 3, shake well, and incubate at 4°C in the dark for 2 hours, shaking every 30 minutes. (Note: The mass ratio of NaBH4 to HRP is 0.08:1.)

[0110] 23. Saturated ammonium sulfate precipitation: Add an equal volume of saturated ammonium sulfate to each tube, incubate at 4°C for 2 h, then centrifuge at 13,000 rpm for 20 min and discard the supernatant.

[0111] 24. Storage: Dissolve the saturated ammonium sulfate precipitate with 20% NBS (newborn calf serum), 500 μL / tube; transfer to a 1.5 ml EP tube; then add 100% imported glycerol, 500 μL / tube, mix thoroughly by pipetting, and store at -20°C. (Note: 20% NBS is 100% newborn calf serum diluted 5-fold with 1 / PBS).

[0112] 25. Solution preparation: (1) Antibody diluent: The formula is as shown in Table 3. Store at 4°C until use. It is not very stable, so do not prepare more than 200 mL at a time. (2) ELISA wash solution (PBST): PBS containing 0.05% Tween-20 (v / v). Store at room temperature until use. (3) Blocking solution: PBS containing 2% (w / v) BSA, pH 7.4. Store at 4°C until use. (4) ELISA stop solution (2 mol / L sulfuric acid): Add 21.7 mL of 98% concentrated sulfuric acid dropwise to 178.3 mL of injection water. Store at 4°C until use.

[0113] Table 3

[0114] 0.1M PB (pH 7.2) 20mL Casein 0.2g BSA 1g Newborn calf serum 5mL Aminopyridine 0.1g glycerin 2mL sucrose 5g Tween-20 1mL Proclin 300 0.1mL pure water 67mL Final volume 95mL

[0115] 25. Dilute the antibodies according to the layout table. Dilute the detection antibody in antibody diluent and the capture antibody in PBS.

[0116] 26. There is no negative control or blank control. The antigen is selected as a monovalent stock solution of matching type.

[0117] 27. Coating: Dilute the capture antibody to 500×, 1000×, 2000×, or 4000× with PBS, according to 100 μl / well as shown in the table. Cover with sealing film and coat overnight at 4°C.

[0118] 28. Washing: Discard the coating solution, add 300 μL / well of PBST (PBS supplemented with 0.05% Tween-20), shake several times, and discard the washing solution. Repeat this three times and pat the plate dry on absorbent paper.

[0119] 29. Blocking: Add 300 μL / well of blocking solution (2% BSA), cover with sealing film, and incubate at 37°C for 2 h.

[0120] 30. Wash the plate 3 times.

[0121] 31. Add antigen: dilute each monovalent stock solution with PBS. The dilution multiple is shown in the layout. Figure 15 The diluted unit price stock solution is Figure 15100 μL / well of the ELISA plate was added, the plate was sealed with film, and incubated at 37°C for 1 hour.

[0122] 32. Wash the plate 3 times.

[0123] 33. Add detection antibody: Dilute the detection antibody to 1000×, 2000×, or 4000× using antibody diluent. Add 100 μL / well to the ELISA plate, cover with sealing film, and incubate at 37°C for 1 hour.

[0124] 34. Wash the plate 5 times.

[0125] 35. Color development: Add 100 μL / well of TMB single-component color development solution, cover with film, and incubate at 37°C in the dark for 15 min.

[0126] 36. Stop: Add 50 μL / well of stop solution (2 M sulfuric acid) and measure the absorbance at 450 nm using a microplate reader.

[0127] The linearity of the standard curves for influenza B virus Bv, influenza B virus By, H1N1 and H3N2 were Figure 11-14 shown.

[0128] Experimental Example 2

[0129] 1. Experimental group: virus maintenance medium prepared in Example 2; control group: XenoTM-S001S serum-free MDCK culture medium.

[0130] 2. Madin Darby Canine Kidney Cells (MDCK-v) (Accession No.: CCTCCNO: C2024173, China Center for Type Culture Collection, Wuhan University, Wuhan, China), influenza viruses Bv, By, H1N1, and H3N2.

[0131] 3. The MDCK-v cell culture conditions are conventional in the art, and the bioreactor parameters are set according to the instructions of the bioreactor manufacturer;

[0132] When MDCK-v cells grow to 10-12×10 6 When the influenza virus maintenance solution of the present invention was used, the influenza virus Bv, By, H1N1, and H3N2 were inoculated respectively, and the control group was cultured in XenoTM-S001S serum-free MDCK medium. The virus titer comparison of the influenza virus maintenance solution of the present invention and the control group cultured in XenoTM-S001S serum-free MDCK medium is shown in Table 4.

[0133] Table 4 Comparison of influenza virus titers (1g (1 / TCID50) / 100μl)

[0134] 12h 24h 48h 72h 96h 120h Experiment Bv 3.1 6.0 7.0 8.2 9.1 8.5 Control Bv 1.9 4.2 5.1 6.1 7.1 6.8 ExperimentBy 3.2 5.8 6.9 7.9 8.9 8.4 By 1.8 3.9 4.8 5.8 7.0 6.0 Experimental H1N1 3.5 5.9 7.0 8.1 9.2 8.7 Control H1N1 2.1 3.7 4.8 5.2 6.9 6.5 Experimental H3N2 3.2 6.1 6.8 7.8 8.8 8.4 Control H3N2 1.7 4.1 5.2 5.1 6.7 6.1

[0135] Compared with the maintenance solution of the control group, the influenza virus maintenance solution provided by the present invention can significantly increase the virus yield. This is because the present invention adds yeast induction culture extract, which not only increases the amount of tryptophan and glutcystine, but also increases other effective ingredients, which can effectively promote the amplification of influenza virus in cells.

[0136] Experimental Example 3

[0137] This experimental example provides a comparison of the hemagglutination titer of influenza virus cultured in influenza virus maintenance medium and DMEM / F-12 basal medium.

[0138] 1. Experimental group: virus maintenance solution prepared in Example 2; control group: DMEM / F-12 basal culture medium.

[0139] 2. Madin Darby Canine Kidney Cells (MDCK-v) (Accession No.: CCTCCNO: C2024173);

[0140] 3. Influenza virus Bv type BVR-26, H1N1 type IVR-215. Set up three parallel assays for each type.

[0141] 4. Cell processing:

[0142] 4.1 Cell Preparation

[0143] 4.1.1 Cells: MDCK-v canine kidney cells.

[0144] 4.1.2 Turn on the electric constant temperature water bath and set the temperature to 37.0℃.

[0145] 4.1.3 Preheat OPM-AM146 culture medium to 37.0°C.

[0146] 4.1.4 Remove one tube of MDCK-v cells from the liquid nitrogen tank and verify the cell name, passage number, batch number, date, density, and other information.

[0147] 4.1.5 Immerse the cell cryopreservation tube vertically in a 37.0℃ electric constant temperature water bath and thaw quickly. Shake rapidly during the water bath and avoid water contact with the tube mouth.

[0148] 4.1.6 After the cells have thawed until only small ice cores remain, thoroughly disinfect the surface of the cryotube with 75.0% ethanol, wipe dry, and place in a biosafety cabinet.

[0149] 4.1.7 Gently disperse the cell suspension in the cryopreserved tube with a 1 μm pipette, sample 50 μL of the cell suspension, and count the cells according to the "Countess3 Cell Counter Use, Cleaning, and Maintenance Standard Operating Procedure" (SOP08018) to determine the viable cell density and cell viability.

[0150] 4.1.8 Take a 15ml centrifuge tube and fill it with 9ml of preheated OPM-AM146 medium. Use a pipette to transfer the cell suspension to the tube and gently pipette to mix. After leveling the tube, centrifuge it at 200 × g for 5 minutes.

[0151] 4.1.9 After centrifugation, discard the supernatant and resuspend and mix in preheated OPM-AM146 medium.

[0152] 4.1.10 Transfer the cell suspension to a T25 / T75 culture flask and add pre-warmed OPM-AM146 medium to the corresponding culture volume.

[0153] 4.1.11 Label the cell batch number, generation number, and passage period, and gently shake the container with both hands to ensure that the cells are evenly distributed in the culture medium.

[0154] 4.1.12 Place the labeled, shaken container in a 37.0°C, 5.0% CO2 cell culture incubator and culture until the cells reach 90% confluence and cover the bottom of the flask. This should last approximately 2-3 days.

[0155] 4.2 Cell passaging

[0156] 4.2.1 Open the biosafety cabinet and turn on the UV lamp for at least 20 minutes.

[0157] 4.2.2 Turn on the electric constant temperature water bath and set the temperature to 37.0℃.

[0158] 4.23 Mix PBS buffer and TrypLE TM Preheat Express and OPM-AM146 culture media to 37.0°C.

[0159] 4.2.4 Before subculturing, observe the cell adhesion in at least four areas under a microscope. Select cells with clear outlines, clear supernatant, and more than 90% confluence as subculturing cells.

[0160] 4.2.5 Disinfect the culture container with 75.0% ethanol and place it in a biosafety cabinet. Discard the cell culture supernatant.

[0161] 4.2.6 Use a pipette to take the corresponding volume of PBS buffer and add it to the culture container. Shake gently to rinse the cell surface and discard. Wash twice in the same way. If it is a multi-layer cell factory, it is necessary to place the factory on its side, raise the sample port side after the liquid level is balanced, place it upright, and then turn it down to a horizontal position to ensure that the liquid is evenly distributed in each layer.

[0162] 4.2.7 Use a pipette to add the appropriate volume of TrypLE pancreatic enzyme to the container to digest the cells. Place the container in a 37.0°C incubator for 20 to 40 minutes. During the digestion process, observe and move the container several times to ensure even heating of the bottom surface.

[0163] 4.2.8 When the cells have been digested into round, shiny spheres and have fallen off the bottom of the container, add the corresponding volume of preheated OPM-AM146 medium to the container to terminate digestion.

[0164] 4.2.9 Gently pipette the cells until they are dispersed into individual pieces and transfer the cell suspension into a centrifuge tube.

[0165] 4.2.10 Balance the centrifuge tube and centrifuge at 200×g for 5 minutes.

[0166] 4.2.11 After centrifugation, discard the supernatant in a biosafety cabinet and add an appropriate volume of pre-warmed OPM-AM146 medium to the centrifuge tube to resuspend the cells. Mix all the cells and gently pipette to evenly distribute the suspension.

[0167] 4.2.12 Take a 50 μL sample of cell suspension and count the cells according to the Standard Operating Procedure for Use, Cleaning, and Maintenance of the Countess3 Cell Counter (SOP08018) to determine the cell density and viability.

[0168] 4.2.13 Select the subculture ratio and culture container according to the experimental requirements, pipette the appropriate cell suspension into each container, and add preheated OPM-AM146 culture medium to each container to the corresponding culture volume.

[0169] 4.2.14 The recommended subculture ratio is 1:5, with subculture every 3 days.

[0170] 4.2.15 Label the cell batch number, passage number, subculture date, and other information, and gently shake the square bottle with both hands to ensure that the cells are evenly distributed. If it is a multi-layer cell factory, place the factory on its side, wait for the liquid level to balance, raise the sample port side, place it upright, and then lower it to a horizontal position to ensure that each layer of cell suspension is evenly distributed. If it is a multi-well plate, shake or move the plate in a figure-8 motion to mix the cells evenly.

[0171] 4.2.16 Place all labeled and shaken containers in an incubator at 37.0℃ and 5.0% CO2 for incubation.

[0172] 5. Virus inoculation:

[0173] 5.1 Before use, add TPCK trypsin to the virus maintenance medium (experimental) and DMEM / F12 (control) to a final concentration of 2 μg / ml.

[0174] 5.2 Pour out the cell culture medium in the culture flask and rinse the cell surface three times with PBS buffer.

[0175] 5.3 Virus strain preparation: Confirm that the strains released are those required for this library construction. Follow the "Bacteria and Virus Strain Management Procedure" (PPC0801) for strain release and complete relevant records. Reconstitute the strains at room temperature and refrigerate until ready for use.

[0176] 5.4 Dilution: Inoculate at an MOI of 0.001. Dilute the virus in virus maintenance medium or DMEM / F12 and inoculate the cell flask. The culture volume is 5 ml.

[0177] 5.5 Virus culture: After inoculation of the virus, place the T25 culture flask in a 37°C, 5% CO2 incubator and culture until the cells are completely drifted, usually 48-96 hours.

[0178] 5.6 Testing: Every 8 hours, collect 100 μL of virus suspension and send it for hemagglutination titer testing. The results for Bv and H1N1 are shown in the table below. Figure 16 and Figure 17 As shown in the figure, it can be seen that the influenza virus cultured using the virus maintenance solution disclosed in the embodiment of the present invention has a higher hemagglutination titer.

[0179] In summary, the virus maintenance solution prepared by the present invention by adding yeast induction culture extract through reasonable proportions can effectively promote the amplification of influenza virus in cells, increase the virus yield, and the hemagglutination titer of the cultured influenza virus is higher.

[0180] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. An influenza virus maintenance solution, characterized in that The virus maintenance solution is composed of a DMEM / F-12 basal culture medium and active ingredients, wherein the active ingredients are composed of amino acid sources, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter is a yeast induction culture extract; the yeast induction culture extract is a YPD liquid culture medium added with L-phenylalanine and citric acid, inoculated with brewer's yeast, cultured for the first time for 12-24 hours, and then glycerol, fennel extract and betaine are added to continue induction culture for 12-24 hours, and the induction culture is terminated. The yeast extract is then extracted; the amount of L-phenylalanine added is 0.2-0.6 g / L, the amount of fennel extract added is 0.5-2 g / L, glycerol is 20-30 g / L, citric acid is 20-40 mM, and betaine is 0.04-0.08 g / L. The yeast extract extraction method comprises adding sodium chloride and protease after the culture is completed; adjusting the pH, adding polysaccharidase after autolysis, and continuing autolysis; inactivating the enzyme, centrifuging to obtain a yeast extract; and vacuum concentrating the yeast extract and spray drying it to obtain a powdered yeast extract. The amino acid sources are L-aspartic acid monohydrate 1-3 mg / L, L-glutamine 50-80 mg / L, L-arginine hydrochloride 10-40 mg / L, L-cystine hydrochloride 4-8 mg / L, L-lysine hydrochloride 16-20 mg / L, L-proline 8-12 mg / L and L-serine 4-8 mg / L; The vitamins are biotin 0.0002-0.0006 mg / L, D-calcium pantothenate 0.2-0.6 mg / L, nicotinamide 0.2-0.6 mg / L, and inositol 2-3 mg / L; The additive is D(+)-galactose 100-300 mg / L; The salts are 5-15 mg / L anhydrous calcium chloride and 200-300 mg / L sodium chloride.

2. A method for preparing the influenza virus maintenance solution according to claim 1, characterized in that: The method comprises: mixing DMEM / F-12 basal culture medium and the active ingredient according to claim 1, and then disinfecting and sterilizing.

3. Use of the influenza virus maintenance solution according to claim 1 in the preparation of influenza virus vaccines.

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