Formula of influenza virus maintenance fluid as well as preparation method and application of influenza virus maintenance fluid

Abstract

The invention discloses an influenza virus maintenance fluid formula as well as a preparation method and application thereof, and particularly relates to the technical field of biological medicines. The virus maintenance liquid comprises a DMEM / F-12 basal culture medium and effective components, the effective components comprise amino acids, vitamins, salts, a virus proliferation accelerant and an additive, and the virus proliferation accelerant comprises a yeast extract. According to the invention, an influenza virus subunit ELISA detection kit is established, and an influenza virus maintenance solution formula is developed on the basis of a DMEM / F-12 basic culture medium by taking an ELISA detection result at a culture endpoint as a response value. The virus yield of the influenza virus on the MDCK-v cell matrix can be obviously improved, and the production cost is reduced.

Description

Technical Field

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

[0002] DMEM / F-12 (DuLbecco's Modified Eagle Medium / Nutrient Mixture F-12) is a widely used basal medium that can be used to support the growth of many different types of mammalian cells after the addition of serum. 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 of DMEM and the 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 virus infection of humans, which can lead to millions of severe cases and hundreds of thousands of deaths each year. It is extremely harmful to human health and has become one of the important public health issues of global concern. At present, human influenza vaccines are mainly used internationally, including subunit vaccines of purified live viruses, inactivated viruses or recombinant viruses. Due to the strong mutation ability of influenza viruses and the influence of factors such as the blocking effect of human antibodies, there are differences between existing vaccine strains and prevalent strains, which reduces the immune effect and protection rate of vaccines. The commonly used method is to use whole genome sequencing technology to determine the antigenicity of newly isolated influenza viruses, and it takes a long time to develop new vaccines based on this.

[0005] The liquid used for virus culture is usually called virus culture medium or virus maintenance medium. Its composition and preparation method directly determine the survival of cells and whether a sufficient number of virus particles with infection and replication ability can be obtained. Virus maintenance medium usually uses serum-free basic culture medium such as DMEM, MEM, DMEM / F-12, M199, 1640, etc. These basic culture media have different effects on influenza virus culture and there is a lot of room for improvement. Summary of the invention

[0006] To this end, the present invention provides a formula of influenza virus maintenance solution 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, which includes a DMEM / F-12 basal culture medium and effective ingredients, wherein the effective ingredients include amino acid sources, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter includes 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] Further, 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, L-serine 6 mg / L.

[0011] Furthermore, the vitamins include biotin, D-calcium pantothenate, nicotinamide, and inositol. As examples, preferably, biotin 0.0002-0.0006 mg / L, D-calcium pantothenate 0.2-0.6 mg / L, nicotinamide 0.2-0.6 mg / L, 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, inositol 2.5 mg / L.

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

[0013] Further, 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 then extracting the 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 extraction method of the yeast extract includes 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; 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 protease are added; the pH is adjusted to 7-8, and polysaccharidase is added after autolysis at 40-50°C for 12-24 hours, 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 spray-dried to obtain a powdered yeast extract; the protease is papain, and the amount used is 500U / mL; the polysaccharidase is amylase, and the amount used is 500U / 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, the method comprising: mixing DMEM / F-12 basal culture medium and effective ingredients to obtain the influenza virus maintenance solution, wherein the effective ingredients include amino acid sources, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter includes yeast induction culture extract.

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

[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 influenza virus maintenance liquid to obtain an influenza virus maintenance liquid that can promote the intracellular amplification of influenza virus, significantly increase the yield of the virus, and at the same time maintain a high titer of the virus, significantly improve the efficiency of the production process, and reduce the production cost;

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

[0025] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0026] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

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

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

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

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

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

[0035] Fig. 9 The rabbit H1N1 peak diagram provided in Experimental Example 1 of the present invention;

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

[0037] Fig.11 The Bv related linear relationship diagram provided in Experimental Example 1 of the present invention;

[0038] Fig.12 The By-related linear relationship diagram provided by Experimental Example 1 of the present invention;

[0039] Fig.13 H1N1 related linear relationship diagram provided in Experimental Example 1 of the present invention;

[0040] Fig.14 H3N2 related linear relationship diagram provided in Experimental Example 1 of the present invention;

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

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

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

[0044] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Except for the raw materials used in the present invention that are separately marked, the other ones are purchased from Merck Biotechnology and McLean's corresponding product 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 normal brewer's yeast culture conditions. The first culture lasted for 12-24 hours, and then glycerol, fennel extract (CAS: 84775-42-8) and betaine were added for continued induction culture for 12-24 hours, and then the culture was continued. After the culture was completed, a 2-5% weight / volume suspension was prepared; 1-3% weight / volume sodium chloride and protease were added; the pH was adjusted to 7-8, and polysaccharidase was added after autolysis at 40-50° C. for 12-24 hours, and the autolysis was continued for 12-24 hours; the enzyme was inactivated, and a yeast extract was obtained by centrifugation; the yeast extract was vacuum concentrated and then spray-dried to obtain a powdered yeast extract; the protease was papain, and the amount was 500 U / mL; the polysaccharidase was amylase, and the amount was 500 U / mL.

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

[0049] The tryptophan content and glutcysteine ​​content 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 glutcysteine ​​increased significantly after induction culture.

[0050] TPCK pancreatic enzyme solution 1g / L: The preparation solvent is 1mM hydrochloric acid, dissolve TPCK pancreatic enzyme (CAS#: LS003741; LOT#: 33P24072), sterilize and filter, prepare at 2-8℃ each time, and store for 6 months; add TPCK pancreatic enzyme solution before subsequent virus culture, with a working concentration of 2μg / ml culture volume.

[0051] Example 1

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

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

[0054] The effect of optimizing the virus maintenance solution formula on virus yield was investigated. The model was a factorial analysis Two-Level8-4IV. The components investigated are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058]

[0059] The 49 ingredients in Table 1 were screened; the concentrations of the ingredients after the initial screening were optimized; and the final determined ingredients 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 Residue 5.87 23 0.2552 Total deviation 22.08 31

[0063] The optimal combination of influenza virus maintenance fluid was finally determined as: 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-calcium pantothenate, niacinamide, 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, nicotinamide 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 medium.

[0068] The DMEM / F-12 basal culture medium and the effective components are mixed and then sterilized 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 medium.

[0073] The DMEM / F-12 basal culture medium and the effective components are mixed and then sterilized 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 medium.

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

[0079] Experimental Example 1

[0080] The influenza virus maintenance solution of Example 2 was used to culture influenza B virus Bv, influenza B virus By, influenza A virus H1N1 and influenza A virus H3N2 (the viruses were all purchased from NIBSC, the same below);

[0081] Establish 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 the subunit antigen of each virus strain. Rabbit serum was used as capture antibody and mouse serum as detection antibody. ELISA detection system was established.

[0083] Rabbit serum and mouse serum: According to the standards, rabbit immune sera and mouse immune sera 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 time, and Freund's incomplete adjuvant for the second and third time. The interval between each immunization is 2 weeks. Inject subcutaneously at multiple points on the back and groin of the rabbit, 1 ml per rabbit, and generally inject 5 to 7 subcutaneous points.

[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 immunization, whole blood was collected from the carotid artery or heart of the four rabbits. The time of blood collection was recorded.

[0090] 7. After blood collection, serum can be separated by placing the blood at 37°C for 30-60 minutes to coagulate. The blood clot is separated from the surface of the collector and placed at 4°C overnight to shrink the blood clot. The blood clot should be suspended as much as possible overnight, and the serum is collected by gravity. The next day, centrifuge at 3000rpm*10min, remove the serum, and water bath at 56°C for 30min.

[0091] 8. After the serum is filtered through a 0.45 μm filter membrane, IgG antibodies are 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 treatment: (1) Cut the dialysis bag into small pieces of appropriate length (10-20cm). (2) Boil the dialysis bag in a large volume of 2% (W / V) sodium bicarbonate and 1mmol / L EDTA (pH8.0) for 10 minutes. (3) Rinse the dialysis bag thoroughly with distilled water. (4) Boil it in 1mmol / L EDTA (pH8.0) for 10 minutes. (5) After cooling, store it at 4 degrees. Make 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 with a 0.22μm or 0.45μm filter membrane before use. (2) Sample preparation: Before loading, ensure that the sample solution has the appropriate ionic strength and pH value. The serum can be diluted 1:1 with binding / washing buffer or added to the binding / washing buffer and dialyzed overnight. (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) Take the Protein G affinity chromatography column out of 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 drain the preservation solution, and continue to flush 5 times the column volume. Place tube A in the binding buffer and tube B in the elution buffer. Set the parameter to "InletA" 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 the chromatography column: Switch the parameter back to "Inlet A" and the system flow rate to 5 mL / min to 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 peak before and after the peak appropriately. Generally, UV80 is used as the boundary, and the peak greater than UV80 is collected. (10) Rinse the chromatography column and pipeline 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-situ 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 [monitored by pH test paper] and mix well. (14) Transfer the antibody to a dialysis bag and dialyze it in 2L PBS buffer at 4°C overnight, changing the dialysis solution every 4 hours for 3 times.

[0098] Rabbit serum purification:

[0099] 13. Prepare the following solutions required 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 treatment: (1) Cut the dialysis bag into small pieces of appropriate length (10-20cm). (2) Boil the dialysis bag in a large volume of 2% (W / V) sodium bicarbonate and 1mmol / L EDTA (pH8.0) for 10 minutes. (3) Rinse the dialysis bag thoroughly with distilled water. (4) Boil it in 1mmol / L EDTA (pH8.0) for 10 minutes. (5) After cooling, store it at 4 degrees. Make 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 with a 0.22μm or 0.45μm filter membrane before use. (2) Sample preparation: Before loading, ensure that the sample solution has the appropriate ionic strength and pH value. The serum can be diluted 1:1 with binding / washing buffer or added to the binding / washing buffer and dialyzed overnight. (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) Take the Protein G affinity chromatography column out of 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 drain the preservation solution, and continue to flush 5 times the column volume. Place tube A in the binding buffer and tube B in the 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 and 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 the chromatography column: Switch the parameter back to "Inlet A" and the system flow rate to 5 mL / min to 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" again to 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 start the program. (9) Observe the result peak graph. When the pH value begins to decrease and the ultraviolet 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, UV80 is used as the boundary, and the peak greater than UV80 is collected. (10) Rinse the chromatography column and pipes 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) 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 [pH test paper monitoring] and mix well. (14) Transfer the antibody to a dialysis bag and dialyze it in 2L PBS buffer at 4℃ overnight, changing the dialysis solution every 4h, and change the solution 3 times.

[0102] 16. Take samples for protein electrophoresis purity analysis and protein content detection. The remaining samples are divided into two portions for freezing. Rabbit serum is divided into several vials of 1 ml and 0.1 ml. Mouse serum is divided into two portions for HRP labeling, a total of four portions. Record the volume and the date of the division.

[0103] HRP labeling

[0104] 17. Label a portion of mouse serum with HRP.

[0105] 18. Prepare carbonate buffer CBS (0.05 M): weigh 1.59 g sodium carbonate and 2.93 g 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 into dialysis bags; 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), add 1.5 ml of ultrapure water to dissolve; (concentration: 20 mg / ml) (2) Weigh 30 mg of NaIO4 (in a 4 ml EP tube), add 1.2 ml of ultrapure water to dissolve; (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, shaking while adding; place the mixed solution at 4°C, away from light for 30 minutes; (4) Take 200 μL of ethylene glycol and add it dropwise to the above solution, shaking while adding, and let it stand at 4°C, away from light for 30 minutes for use; (stop activation). (The volume ratio of HRP: NaIO4: ethylene glycol is 5:4:1)

[0108] 21. Antibody-HRP coupling: Take 200 μL of activated HRP and add it to the above dialysis bags containing monoclonal antibodies, dialyze with CB, change the solution once every 2 hours (3 times), and dialyze overnight at 4℃ in the dark; then collect the solution in the dialysis bag with 4ml EP tubes. (The mass ratio of Ab:HRP:NaIO4 is 1:1:1).

[0109] 22. Stop the reaction: Prepare NaBH4: add 2ml ultrapure water to a 4ml EP tube; weigh 4mg NaBH4 and dissolve it in the EP tube (keep away from light); add 80μL NaBH4 solution to the EP tube in step 3, shake well, and stand at 4℃ away from light for 2h, shaking every 30min. (Note: The mass ratio of NaBH4:HRP is 0.08:1)

[0110] 23. Saturated ammonium sulfate precipitation: add an equal volume of saturated ammonium sulfate to each tube, let stand at 4°C for 2 hours, then centrifuge at 13000 rpm for 20 minutes, and discard the supernatant;

[0111] 24. Storage: Dissolve the saturated ammonium sulfate precipitate with 20% NBS (newborn calf serum), 500μL / tube; transfer to a 1.5ml EP tube; then add 100% imported glycerol, 500μL / tube, mix well by blowing, and store at -20℃. (Note: 20% NBS means diluting 100% newborn calf serum 5 times with 1*PBS).

[0112] 25. Solution preparation: (1) Antibody diluent: The formula is as shown in Table 3. Store at 4℃ for future use. It is not very stable. Do not prepare more than 200mL at a time. (2) ELISA wash solution (PBST): PBS containing 0.05% Tween-20 (v / v). Store at room temperature for future use. (3) Blocking solution: PBS containing 2% (w / v) BSA, pH 7.4. Store at 4℃ for future use. (4) ELISA stop solution (2mol / L sulfuric acid): Add 21.7mL of 98% concentrated sulfuric acid dropwise to 178.3mL of injection water. Store at 4℃ for future 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 with antibody diluent and the capture antibody with 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×, 4000× with PBS, according to 100μl / well in the table, cover with sealing film, and coat overnight at 4℃.

[0118] 28. Washing: shake off the coating solution, add PBST (PBS with 0.05% Tween-20) at 300 μL / well, shake several times and then shake off the washing solution. Repeat 3 times and pat the ELISA 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 hours.

[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. Fig.15 The diluted unit price stock solution is Fig.15100 μL / well of the ELISA plate was added, the plate was covered with a sealing film, and incubated at 37°C for 1 h.

[0122] 32. Wash the plate 3 times.

[0123] 33. Add detection antibody: Use antibody diluent to dilute the detection antibody to 1000×, 2000×, 4000×, 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 sealing film, and develop color at 37℃ in the dark for 15 minutes.

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

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

[0128] Experimental Example 2

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

[0130] 2. Canine kidney cells MDCK-v (Madin Darby Canine Kidney Cells, deposit number: CCTCCNO: C2024173, deposit address: China Center for Type Culture Collection, Wuhan University, Wuhan, China), influenza viruses Bv, By, H1N1, H3N2.

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

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

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

[0134] 12h 24h 48h 72h 96h 120h Experiment Bv 3.1 6.0 7.0 8.2 9.1 8.5 Compare 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 a 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 viruses 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. Canine kidney cells MDCK-v (Madin Darby Canine Kidney Cells, deposit number: CCTCCNO: C2024173,)

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

[0141] 4. Cell processing:

[0142] 4.1 Cell preparation

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

[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 medium to 37.0℃.

[0146] 4.1.4 Take out a tube of MDCK-v cells from the liquid nitrogen tank and check the cell name, generation, batch number, date, density number and other information.

[0147] 4.1.5 Immerse the cell cryopreservation tube vertically in a 37.0℃ electric constant temperature water bath for rapid thawing. Shake rapidly during the water bath and avoid water contacting the tube mouth.

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

[0149] 4.1.7 Use a 1m pipette to gently disperse the cell suspension in the cryopreservation tube, sample 50μL of the cell suspension, count the cells according to the "Standard Operating Procedures for Use, Cleaning and Maintenance of Countess3 Cell Counter" (SOP08018), and detect the live cell density and cell viability.

[0150] 4.1.8 Take a 15ml centrifuge tube, fill it with 9ml preheated OPM-AM146 culture medium, transfer the cell suspension to the centrifuge tube with a pipette, and gently pipette to mix. After the centrifuge tube is balanced, place it in a centrifuge and centrifuge 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 Mark the cell batch number, generation number, subculture period and other information, 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 and shaken container in a 37.0℃, 5.0% CO2 cell culture incubator and culture until the cells reach 90% confluence and cover the bottom of the bottle. The culture period is about 2 to 3 days.

[0155] 4.2 Cell passaging

[0156] 4.2.1 Open the biosafety cabinet and turn on the UV light for more than 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 medium to 37.0°C.

[0159] 4.2.4 Before subculturing, observe the cell adhesion in at least four areas under a microscope, and 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, discarding 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 it. Wash twice in the same way. If it is a multi-layer cell factory, 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 each layer of liquid is evenly distributed.

[0162] 4.2.7 Use a pipette to take a corresponding volume of TrypLE pancreatic enzyme and add it to the container to digest the cells. Place the container in a 37.0℃ incubator for digestion. The digestion time is 20min to 40min. During this period, observe and move the container several times to ensure uniform heating of the bottom surface.

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

[0164] 4.2.9 Gently pipette the cells until they are dispersed into a single state 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 the biosafety cabinet, take an appropriate volume of preheated OPM-AM146 medium and add it to the centrifuge tube to resuspend the cells. Mix all the cells and gently pipette evenly.

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

[0168] 4.2.13 According to the experimental requirements, select the subculture ratio and culture container, 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 Mark the cell batch number, generation number, culture 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, it is necessary to place the factory on its side, raise the sample port side after the liquid level is balanced, and then put it upright and then turn it down to a horizontal position to ensure that each layer of cell suspension is evenly distributed. If it is a multi-well plate, oscillate or use an 8-shaped trajectory to mix to ensure that the cells are evenly distributed.

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

[0172] 5. Virus inoculation:

[0173] 5.1 TPCK trypsin was added to the virus maintenance medium (experimental) and DMEM / F12 (control) before use to make the final concentration of TPCK trypsin 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 Preparation of virus strains: Confirm that the virus strains to be released are those required for the current library construction. The virus strains to be released shall be executed in accordance with the "Management Procedures for Bacteria and Viruses" (PPC0801), and relevant records shall be filled in. The virus strains shall be reconstituted at room temperature and refrigerated for later use.

[0176] 5.4 Dilution: Inoculate at 0.001 MOI. Dilute the virus with virus maintenance solution or DMEM / F12 and inoculate into the cell bottle. The culture volume is 5 ml.

[0177] 5.5 Virus culture: After inoculation of the virus, the T25 culture flask is placed in a 37°C, 5% CO2 incubator and cultured 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 of Bv and H1N1 are shown in Fig.16 and Fig.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 present invention adds a virus maintenance solution prepared by a yeast induction culture extract through a reasonable ratio, which can effectively promote the amplification of influenza viruses in cells, increase the virus yield, and the hemagglutination titer of the cultured influenza viruses is higher.

[0180] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. An influenza virus maintenance solution, characterized in that: The virus maintenance solution comprises a DMEM / F-12 basal culture medium and effective ingredients, wherein the effective ingredients include amino acid sources, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter comprises a yeast induction culture extract.

2. An influenza virus maintenance solution according to claim 1, characterized in that: The amino acid sources include L-aspartic acid monohydrate, L-glutamine, L-arginine hydrochloride, L-cystine hydrochloride, L-lysine hydrochloride, L-proline and L-serine.

3. An influenza virus maintenance solution according to claim 1, characterized in that: The vitamins include biotin, calcium D-pantothenate, niacinamide, and inositol.

4. An influenza virus maintenance solution according to claim 1, characterized in that: The salts include anhydrous calcium chloride and sodium chloride.

5. The influenza virus maintenance solution according to claim 1, characterized in that: The additive includes D(+)-galactose.

6. The influenza virus maintenance solution according to claim 1, characterized in that: The yeast induction culture extract is prepared by adding L-phenylalanine and citric acid to a YPD liquid culture medium and then inoculating brewer's yeast, culturing for 12-24 hours for the first time, then adding glycerol, fennel extract and betaine to continue induction culture for 12-24 hours, and extracting the yeast extract after the induction culture is completed.

7. An influenza virus maintenance solution according to claim 6, characterized in that: 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.

8. An influenza virus maintenance solution according to claim 6, characterized in that: The method for extracting the yeast extract comprises adding sodium chloride and protease after the culture is completed; adjusting the pH, adding polysaccharide enzyme after autolysis, and continuing the autolysis; inactivating the enzyme, and centrifuging to obtain a yeast extract; vacuum concentrating the yeast extract, and then spray drying to obtain a powdered yeast extract.

9. A method for preparing an influenza virus maintenance solution according to any one of claims 1 to 8, characterized in that: The method comprises: mixing DMEM / F-12 basic culture medium and effective components, wherein the effective components include amino acid sources, vitamins, salts, virus proliferation promoters and additives, wherein the virus proliferation promoter includes yeast induction culture extract.

10. Use of an influenza virus maintenance solution in the preparation of influenza virus vaccines.

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