Avian influenza virus culture method

Abstract

The invention provides a culture method of avian influenza virus, and relates to the technical field of microbe amplification culture. The culture method of the avian influenza virus comprises the following steps: performing cell culture on host cells in a reactor to obtain a culture solution A, then inoculating avian influenza cytovirus in the culture solution A to infect the host cells, then performing virus infection culture, and obtaining an avian influenza virus supernatant after amplification culture after cytopathy; wherein the inoculation volume of the avian influenza cell virus solution is 0.05-0.06% of the volume of the culture solution A. According to the culture method, the avian influenza virus culture process, virus inoculation volume setting and other processes are adjusted, and research verifies that the virus titer of avian influenza virus culture can be effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of microbial expansion culture, in particular to a method for culturing avian influenza virus. Background Art

[0002] Avian influenza virus is a viral influenza of poultry. It is an infectious disease caused by influenza A virus, ranging from respiratory system to severe systemic sepsis. Once an avian influenza virus breaks out, it often leads to the death of a large number of poultry, causing serious losses to the breeding industry. Therefore, the development of a vaccine against avian influenza virus has become very necessary and urgent.

[0003] In the development process of existing avian influenza virus vaccines, there are certain problems in virus replication and amplification culture. That is, due to the capacity limitation of the cell reactor (usually a 50L cell reactor is used), the inoculation density and inoculation volume of host cells and viruses are not easy to control, which usually leads to an excessively large old liquid ratio, which is not conducive to virus replication and amplification culture.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a method for culturing avian influenza virus. The method of the present invention can obtain a better virus titer, providing a basis for the research of avian influenza virus and the preparation of vaccine.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The present invention provides a method for culturing avian influenza virus, the method comprising:

[0008] The host cells are cultured in a reactor to obtain a culture fluid A, and then the avian influenza virus is inoculated into the culture fluid A to infect the host cells, and then the virus infection culture is performed, and after the cell pathological changes occur, the avian influenza virus supernatant after amplification culture is obtained;

[0009] During the inoculation, the inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A.

[0010] The strain of the avian influenza virus is a H9 subtype avian influenza virus strain.

[0011] Furthermore, the cell culture and virus infection culture are both carried out in a reactor;

[0012] Preferably, the reactor is a 50L cell culture reactor.

[0013] Furthermore, the culture medium in the cell culture process is serum-free SF003 culture medium;

[0014] And / or, the culture medium during the virus infection culture process is serum-free SF003 culture medium.

[0015] Furthermore, the host cell is an MDCK cell.

[0016] Furthermore, during the cell culture, the initial cell density of the host cells is: 0.25-1.5×10 6 cells / mL;

[0017] When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.2×10 6 cells / mL.

[0018] Furthermore, the temperature of the virus infection culture is 33-35°C, preferably 33°C.

[0019] Furthermore, the pH value during the virus infection culture process is 6.8-7.2.

[0020] Furthermore, the culture method comprises:

[0021] (A) Cell culture: 0.25~1.5×10 6 MDCK cells were inoculated in serum-free SF003 medium and cultured until the cell density reached 7-16×10 6 cells / mL;

[0022] Serum-free SF003 medium was then added to adjust the cell density to 2.5-3.2×10 6 cells / mL, and culture medium A was obtained;

[0023] (B) Virus inoculation: TPCK-trypsin was added to culture medium A, and then the avian influenza virus venom was inoculated into culture medium A;

[0024] The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A;

[0025] (C) Virus culture: The culture medium inoculated in step (B) was cultured at 33° C. and pH 6.8-7.2 for 48-72 h until the HA result was ≥11Log2, and the supernatant was collected.

[0026] Furthermore, the amount of TPCK-pancreatin added in step (B) is 5 μg / mL;

[0027] And / or, the step (B) further comprises a feeding step after the inoculation, wherein the feeding medium of the feeding is a high-nutrient concentrated medium;

[0028] And / or, the culture medium after inoculation in step (C) further comprises a second feeding step after culturing for 24 hours, and the culture medium for the second feeding is the same as the feeding medium in step (B).

[0029] Furthermore, the titer of the virus supernatant in step (C) is 9.5 to 10.0 Log (TCID 50 / mL).

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The avian influenza virus culture method provided by the present invention comprises the steps of culturing host cells in a reactor to obtain a culture solution A, then inoculating avian influenza virus in the culture solution A to infect host cells, and then carrying out virus infection culture, and obtaining an avian influenza virus supernatant after amplification culture after cell pathological changes; wherein the inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture solution A. The present application can effectively improve the virus titer of the existing H9 subtype avian influenza virus strain culture through the avian influenza virus culture process and the setting of the virus inoculation volume, as verified by research. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0033] According to one aspect of the present invention, a method for culturing avian influenza virus comprises:

[0034] The host cells are cultured in a reactor to obtain a culture fluid A, and then the avian influenza virus is inoculated into the culture fluid A to infect the host cells, and then the virus infection culture is performed, and after the cell pathological changes occur, the avian influenza virus supernatant after amplification culture is obtained;

[0035] During the inoculation, the inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A.

[0036] The strain of the avian influenza virus is a H9 subtype avian influenza virus strain.

[0037] The avian influenza virus culture method provided by the present invention comprises the steps of culturing host cells in a reactor to obtain a culture solution A, then inoculating avian influenza virus in the culture solution A to infect host cells, and then carrying out virus infection culture, and obtaining an avian influenza virus supernatant after amplification culture after cell pathological changes; wherein the inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture solution A. The present application can effectively improve the virus titer of the existing H9 subtype avian influenza virus culture through the avian influenza virus culture process and the setting of the virus inoculation volume, as verified by research.

[0038] In a preferred embodiment of the present invention, the cell culture and virus infection culture are both carried out in a reactor;

[0039] Preferably, the reactor is a 50L cell culture reactor.

[0040] In a preferred embodiment of the present invention, the culture medium in the cell culture process is serum-free SF003 culture medium;

[0041] And / or, the culture medium during the virus infection culture process is serum-free SF003 culture medium.

[0042] In a preferred embodiment of the present invention, the host cell is an MDCK cell.

[0043] In the above preferred embodiment, the initial cell density of the host cells during cell culture is: 0.25-1.5×10 6 cells / mL;

[0044] When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.2×10 6 cells / mL.

[0045] In a preferred embodiment of the present invention, the temperature of the virus infection culture is 33-35°C, preferably 33°C.

[0046] In a preferred embodiment of the present invention, the pH value during the virus infection culture process is 6.8-7.2.

[0047] In a preferred embodiment of the present invention, the culturing method comprises:

[0048] (A) Cell culture: 0.25~1.5×10 6 MDCK cells were inoculated in serum-free SF003 medium and cultured until the cell density reached 7-16×10 6 cells / mL;

[0049] Serum-free SF003 medium was then added to adjust the cell density to 2.5-3.2×10 6 cells / mL, and culture medium A was obtained;

[0050] (B) Virus inoculation: TPCK-trypsin was added to culture medium A, and then the avian influenza virus venom was inoculated into culture medium A;

[0051] The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A;

[0052] (C) Virus culture: The culture medium inoculated in step (B) was cultured at 33° C. and pH 6.8-7.2 for 48-72 h until the HA result was ≥11Log2, and the supernatant was collected.

[0053] In a preferred embodiment of the present invention, the amount of TPCK-pancreatin added in step (B) is 5 μg / mL;

[0054] And / or, the step (B) further comprises a feeding step after the inoculation, wherein the feeding medium of the feeding is a high-nutrient concentrated medium;

[0055] And / or, the culture medium after inoculation in step (C) further comprises a second feeding step after culturing for 24 hours, and the culture medium for the second feeding is the same as the feeding medium in step (B).

[0056] In a preferred embodiment of the present invention, the titer of the virus supernatant in step (C) is 9.5-10.0 Log (TCID 50 / mL).

[0057] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0058] The following examples illustrate the method for culturing avian influenza virus of the present application by taking a 50L cell reactor as an example.

[0059] Example 1

[0060] A method for culturing avian influenza virus, comprising:

[0061] (1) Inoculate MDCK cells into a 50 L cell reactor;

[0062] The cell culture medium was serum-free SF003 medium, and the seeding density of MDCK cells was 0.25-1.5×10 6 cells / mL, cell culture volume is 16-20L, rotation speed is 25-30rpm;

[0063] MDCK cells were cultured at 37°C until the third day after inoculation, when the cell density reached 7-16×10 6 cells / mL, add serum-free SF003 medium to adjust the cell density to 2.5-3.2×10 6 cells / mL, and culture medium A was obtained;

[0064] (2) adding 5 μg / mL TPCK-trypsin to culture medium A, then supplementing feed A to the volume of culture medium A, and inoculating venom containing H9 subtype avian influenza virus strain into culture medium A;

[0065] The feed A is a nutrient concentrated culture medium, specifically the NF01d culture medium produced by Zhejiang Yishengke Biotechnology Co., Ltd.

[0066] The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A;

[0067] (3) The temperature of the culture medium in step (2) is adjusted to 33°C for virus culture; when the virus culture reaches 24 hours, supplement the culture medium A with 1% of the volume of feed A, introduce 0.01-0.1VVM of compressed air, and adjust 0.01-0.1VVM according to the changes of dissolved oxygen and pH during the culture, dissolved oxygen 40%, pH 6.8-7.2; when the pH is lower than 6.9, the reactor automatically controls the flow to add 0.9mol / L sodium bicarbonate solution. Culture for 48-72 hours, take samples every 24 hours during the culture process to detect the density and viability of live cells, determine the glucose and lactic acid content, osmotic pressure and sample HA, and harvest when the HA result is ≥11Log2.

[0068] Example 2

[0069] A method for culturing avian influenza virus, comprising:

[0070] (1) Inoculate MDCK cells into a 50 L cell reactor;

[0071] The cell culture medium was serum-free SF003 medium, and the seeding density of MDCK cells was 1.0-1.3×10 6 cells / mL, cell culture volume is 16-20L, rotation speed is 25-30rpm;

[0072] MDCK cells were cultured at 37°C until the third day, when the cell density reached 4.0-6.0×10 6 cells / mL, and culture medium A was obtained;

[0073] (2) Same as Example 1.

[0074] (3) Same as Example 1.

[0075] Example 3

[0076] A method for culturing avian influenza virus, comprising:

[0077] (1) Inoculate MDCK cells into a 50 L cell reactor;

[0078] The cell culture medium was serum-free SF003 medium, and the seeding density of MDCK cells was 0.25-1.5×10 6 cells / mL, cell culture volume is 16-20L, rotation speed is 25-30rpm;

[0079] MDCK cells were cultured at 37°C until the third day after inoculation, when the cell density reached 7-16×10 6 cells / mL, add serum-free SF003 medium to adjust the cell density to 3.5-4.5×10 6 cells / mL, and culture medium A was obtained;

[0080] (2) Same as Example 1.

[0081] (3) Same as Example 1.

[0082] In this example, in addition to step (1) adding serum-free SF003 culture medium to adjust the cell density to 3.5-4.5×10 6 Except for "cells / mL", the rest is the same as Example 1.

[0083] Example 4

[0084] A method for culturing avian influenza virus, comprising:

[0085] (1) Inoculate MDCK cells into a 50 L cell reactor;

[0086] The cell culture medium was serum-free SF003 medium, and the seeding density of MDCK cells was 0.25-1.5×10 6 cells / mL, cell culture volume is 16-20L, rotation speed is 25-30rpm;

[0087] MDCK cells were cultured at 37°C until the third day after inoculation, when the cell density reached 7-16×10 6 cells / mL, add serum-free SF003 medium to adjust the cell density to 1.5-2.0×10 6 cells / mL, and culture medium A was obtained;

[0088] (2) Same as Example 1.

[0089] (3) Same as Example 1.

[0090] In this example, in addition to adding serum-free SF003 culture medium to adjust the cell density to 1.5-2.0×10 6 Except for "cells / mL", the rest is the same as Example 1.

[0091] Example 5

[0092] A method for culturing avian influenza virus, comprising:

[0093] (1) Same as Example 1.

[0094] (2) Add 5 μg / mL TPCK-trypsin to culture medium A, then supplement with feed B (lipid virus enhancer), and inoculate the venom containing the H9 subtype avian influenza virus strain into culture medium A;

[0095] The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A;

[0096] The feed B is the E1 additive produced by Shanghai Duoning Biotechnology Co., Ltd.

[0097] (3) Same as Example 1.

[0098] Example 6

[0099] A method for culturing avian influenza virus, comprising:

[0100] (1) Same as in Example 1;

[0101] (2) Same as in Example 1;

[0102] (3) adjusting the temperature of the culture solution in step (2) to 33°C for virus culture;

[0103] When the virus is cultured for 24 hours, supplement C (nucleoside virus enhancer) with 1% of the volume of culture medium A, and introduce 0.01-0.1VVM of compressed air. According to the changes of dissolved oxygen and pH in the culture, 0.01-0.1VVM is adjusted, dissolved oxygen is 40%, pH is 6.8-7.2, and when the pH is lower than 6.9, the reactor automatically controls the flow to add 0.9mol / L sodium bicarbonate solution. Cultivate for 48-72 hours, and take samples every 24 hours during the cultivation process to detect the density and viability of living cells, determine the content of glucose and lactic acid, osmotic pressure and sample HA, and harvest when the HA result is ≥11Log2.

[0104] The feed C is the E4 additive produced by Shanghai Duoning Biotechnology Co., Ltd.

[0105] Example 7

[0106] A method for culturing avian influenza virus, comprising:

[0107] (1) Same as Example 1.

[0108] (2) Add 5 μg / mL TPCK-trypsin to culture medium A, and then inoculate the venom containing the H9 subtype avian influenza virus strain into the culture medium A;

[0109] The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A;

[0110] (3) Same as Example 1.

[0111] This embodiment is the same as embodiment 1 except that the type of feed in step (2) is different.

[0112] Example 8

[0113] A method for culturing avian influenza virus, comprising:

[0114] (1) Same as in Example 1;

[0115] (2) Same as in Example 1;

[0116] (3) adjusting the temperature of the culture solution in step (2) to 33°C for virus culture;

[0117] When the virus is cultured for 24 hours, 0.01-0.1VVM of compressed air is introduced, and 0.01-0.1VVM is adjusted according to the changes in dissolved oxygen and pH during the culture, with dissolved oxygen of 40%, pH 6.8-7.2, and when the pH is lower than 6.9, the reactor automatically controls the flow to add 0.9mol / L sodium bicarbonate solution. Cultivate for 48-72 hours, and take samples every 24 hours during the culture process to detect the density and viability of living cells, determine the content of glucose and lactic acid, osmotic pressure and sample HA, and harvest when the HA result is ≥11Log2.

[0118] This example is the same as Example 1 except that the type of "feeding when the virus is cultured for 24 hours" in step (3) is different.

[0119] Example 9

[0120] This example is the same as Example 1 except that the temperature of virus culture in step (3) is 35°C.

[0121] Example 10

[0122] This example is the same as Example 1 except that the temperature of virus culture in step (3) is 31°C.

[0123] Embodiment 11

[0124] This example is the same as Example 1 except that the pH value of the virus culture in step (3) is 6.3-6.5.

[0125] Example 12

[0126] This example is the same as Example 1 except that the pH value of the virus culture in step (3) is 7.5-7.8.

[0127] Comparative Example 1

[0128] This comparative example is the same as Example 1 except that in step (2), "the inoculation volume of the avian influenza virus venom is 0.005 to 0.006% of the volume of the culture medium A".

[0129] Comparative Example 2

[0130] This comparative example is the same as Example 1 except that in step (2), "the inoculation volume of the avian influenza virus venom is 0.03-0.04% of the volume of the culture medium A".

[0131] Comparative Example 3

[0132] This comparative example is the same as Example 1 except that in step (2), "the inoculation volume of the avian influenza virus venom is 0.5-0.6% of the volume of the culture medium A".

[0133] Test Example 1

[0134] In order to show that the avian influenza virus culture method of the present application can effectively improve the virus titer of the H9 subtype avian influenza virus strain culture, the virus supernatants prepared in Examples 1 to 12 and Comparative Examples 1 to 3 were subjected to HA (hemagglutination test) and virus titer tests, and the specific results are shown in Table 1.

[0135] Table 1:

[0136]

[0137]

[0138]

[0139] As can be seen from the above, the virus supernatant prepared in Example 1 of the present application has a higher hemagglutination result, HA11Log2, and a higher virus titer, with a virus titer of 9.5-10.0Log (TCID 50 / mL);

[0140] In Example 2, the seeding density of MDCK cells was only 1.0-1.3×10 6 cells / mL, and the cell density was only 4.0-6.0×10 6cells / mL, the cell density cannot reach 7~16×10 6 cells / mL. If serum-free SF003 medium is added to dilute the culture medium, the old liquid ratio will be too large in the later virus culture process, and the effect of virus expansion culture will be relatively poor.

[0141] Example 3 The initial cell density of virus culture is 3.5-4.5×10 6 cells / mL, the cell density is high, the nutrients in medium A and feed A cannot support high-density virus culture, and the effect of virus expansion culture is relatively poor;

[0142] Example 4 The initial cell density of virus culture is 1.5-2.0×10 6 cells / mL, there is not enough cell number to support virus replication, resulting in low HA and virus titer results;

[0143] Example 5 The inoculation process does not include the step of feeding. Feed B is added to the virus culture. First, the cell growth process cannot reach a cell density of 7 to 16×10 6 cells / mL, the nutrients in feed B cannot meet the maintenance of cell growth and viral replication;

[0144] Example 6: Feed A was added during the inoculation process. When the virus was cultured for 24 hours, feed C was added. The nutritional components of feed C could not meet the maintenance of cell growth and virus replication.

[0145] Example 7 When the virus was cultured for 24 hours, feed A was added. First, the cell growth process could not reach a cell density of 7 to 16×10 6 cells / mL, feed A was added during the virus culture phase, but there were not enough cells to replicate the virus, resulting in lower HA and virus titer results;

[0146] Example 8 No feeding was performed when the virus culture reached 24 hours, and there was not enough nutrition to maintain cell growth and virus replication during the virus culture;

[0147] In Example 9, the virus culture temperature was 35°C, which was not enough to maintain virus replication; in Example 10, the virus culture temperature was 31°C, which was not enough to maintain normal cell growth and affected virus replication;

[0148] The pH value of the virus culture in Example 11 is 6.3-6.5, and the acidic environment cannot maintain normal cell growth, affecting virus replication; the pH value of the virus culture in Example 12 is 7.5-7.8, and the alkaline environment cannot maintain normal cell growth, affecting virus replication;

[0149] In Comparative Example 1, the inoculation volume of the avian influenza cell virus venom was 0.005-0.006% of the volume of the culture medium A, the inoculation amount was too low, and the HA and virus titers were unqualified. In Comparative Example 2, the inoculation volume of the avian influenza cell virus venom was 0.03-0.04% of the volume of the culture medium A, which met the seedling standard, but was not the experimental condition with the best results. In Comparative Example 3, the inoculation volume of the avian influenza cell virus venom was 0.5-0.6% of the volume of the culture medium A. According to the characteristics of the avian influenza strain, the higher the inoculation amount, the lower the HA.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for culturing avian influenza virus, characterized in that: The culture method comprises: The host cells are cultured in a reactor to obtain a culture fluid A, and then the avian influenza virus is inoculated into the culture fluid A to infect the host cells, and then the virus infection culture is performed, and after the cell pathological changes occur, the avian influenza virus supernatant after amplification culture is obtained; During the inoculation, the inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A. The strain of the avian influenza virus is a H9 subtype avian influenza virus strain.

2. The method for culturing avian influenza virus according to claim 1, characterized in that: The cell culture and virus infection culture are both carried out in a reactor; Preferably, the reactor is a 50L cell culture reactor.

3. The method for culturing avian influenza virus according to claim 1, characterized in that: The culture medium in the cell culture process is serum-free SF003 culture medium; And / or, the culture medium during the virus infection culture process is serum-free SF003 culture medium.

4. The method for culturing avian influenza virus according to claim 1, characterized in that: The host cell is MDCK cell.

5. The method for culturing avian influenza virus according to claim 4, characterized in that: During the cell culture, the initial cell density of the host cells is: 0.25-1.5×10 6 cells / mL; When the virus is infected and cultured, the initial cell density of the host cells is 2.0 to 3.2×10 6 cells / mL.

6. The method for culturing avian influenza virus according to claim 1, characterized in that: The temperature of the virus infection culture is 33-35°C, preferably 33°C.

7. The method for culturing avian influenza virus according to claim 1, characterized in that: The pH value during the virus infection culture process is 6.8-7.

2.

8. The method for culturing avian influenza virus according to claim 1, characterized in that: The culture method comprises: (A) Cell culture: 0.25~1.5×10 6 MDCK cells were inoculated in serum-free SF003 medium and cultured until the cell density reached 7-16×10 6 cells / mL; Serum-free SF003 medium was then added to adjust the cell density to 2.5-3.2×10 6 cells / mL, and culture medium A was obtained; (B) Virus inoculation: TPCK-trypsin was added to culture medium A, and then the avian influenza virus venom was inoculated into culture medium A; The inoculation volume of the avian influenza virus venom is 0.05-0.06% of the volume of the culture medium A; (C) Virus culture: The culture medium inoculated in step (B) was cultured at 33° C. and pH 6.8-7.2 for 48-72 h until the HA result was ≥11Log2, and the supernatant was collected.

9. The method for culturing avian influenza virus according to claim 8, characterized in that: The amount of TPCK-pancreatin added in step (B) is 5 μg / mL; And / or, the step (B) further comprises a feeding step after the inoculation, wherein the feeding medium of the feeding is a high-nutrient concentrated medium; And / or, the culture medium after inoculation in step (C) further comprises a second feeding step after culturing for 24 hours, and the culture medium for the second feeding is the same as the feeding medium in step (B).

10. The method for culturing avian influenza virus according to claim 8, characterized in that: The titer of the virus supernatant in step (C) is 9.5-10.0 Log (TCID 50 / mL).

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