Respiratory syncytial virus culture medium, respiratory syncytial virus antigen as well as preparation method and application of respiratory syncytial virus antigen

Abstract

The invention relates to the technical field of biology, in particular to a respiratory syncytial virus culture medium, a respiratory syncytial virus antigen and a preparation method and application of the respiratory syncytial virus antigen. The respiratory syncytial virus antigen obtained by the respiratory syncytial virus culture medium and the preparation method is high in activity, is suitable for large-scale production, and can be used for preparing respiratory syncytial virus vaccines; and / or preparing a reagent and / or a kit for detecting the anti-respiratory syncytial virus specific IgM antibody, thereby laying a foundation for the development of RSV vaccines and the detection of serological specific antibodies.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a respiratory syncytial virus culture medium, a respiratory syncytial virus antigen, and a preparation method and application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide, resulting in significant morbidity and considerable mortality. In the elderly, RSV manifests as recurrent infections leading to fever, pneumonia, and even death.

[0003] RSV is a negative-strand RNA virus belonging to the family Paramyxoviridae. A and B are the two main subtypes of RSV. Although strains of both subtypes often infect at the same time, only type A or type B often dominates the infection. RSV is an enveloped virus with a virus particle diameter of 100~300 nm and a genome length of about 15.2 kb. It contains 10 genes encoding 11 proteins, of which two transmembrane proteins G protein and F protein are the two main protective antigens of the virus.

[0004] Epidemiological surveys of respiratory pathogens in some regions show that the infection rate in the population is very high, with about 90% of adults infected. Although antibodies with neutralizing and complement-binding abilities can be produced after infection with RSV, reinfection is still common. Anti-RSV-specific IgM antibodies can appear as early as about one week after onset and can persist for 2 to 3 months.

[0005] Respiratory syncytial virus is one of the most important viral pathogens of severe respiratory infections in infants and young children, with a wide prevalence and high incidence. WHO has listed RSV vaccine as one of the priority vaccines for development, but there are many problems, such as difficulty in RSV tissue culture, lack of experimental animal models, and immunopathology and safety issues caused by vaccines. In addition, the virus has poor stability, which is generally lower than influenza virus and parainfluenza virus, and poor tolerance to various environmental factors. Low pH, heat, common disinfectants, organic solvents and other environments can inactivate it very quickly. Respiratory syncytial virus is difficult to culture, which in turn limits the progress of clinical research and development of RSV vaccine. Summary of the invention

[0006] In view of this, the present invention provides a respiratory syncytial virus culture medium, a respiratory syncytial virus antigen, and a preparation method and application thereof. The respiratory syncytial virus antigen obtained by using the respiratory syncytial virus culture medium and preparation method of the present invention has high activity, is suitable for large-scale production, and can be used to prepare a respiratory syncytial virus vaccine; and / or prepare a reagent and / or kit for detecting anti-respiratory syncytial virus specific IgM antibodies, laying a foundation for the development of RSV vaccines and serological specific antibody detection.

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

[0008] The present invention provides a respiratory syncytial virus culture medium, wherein each liter of the culture medium comprises the following components:

[0009] 12~14g DMEM medium, 4~6g Hepes, 2~3g NaHCO 3 , 0.1~0.2 g glutamine, 0.1~0.2 g sodium pyruvate, 2~8 μL β-mercaptoethanol, 100KU penicillin, 0.1g streptomycin, and the balance is water.

[0010] In some specific embodiments of the present invention, the culture medium further comprises 5% by volume of newborn calf serum.

[0011] The present invention also provides the use of the respiratory syncytial virus culture medium in any of the following items:

[0012] (I) culture of respiratory syncytial virus; and / or

[0013] (II) Preparation of respiratory syncytial virus antigen.

[0014] The invention also provides a method for culturing respiratory syncytial virus, comprising culturing respiratory syncytial virus by using the respiratory syncytial virus culture medium.

[0015] The present invention also provides a method for preparing a respiratory syncytial virus antigen, comprising the following steps:

[0016] Step 1, cell preparation: culturing host cells to adhere to the wall to obtain adherent host cells;

[0017] Step 2, virus inoculation: mixing the respiratory syncytial virus and the respiratory syncytial virus culture medium, and inoculating the mixture into the adherent host cells for culture to obtain a respiratory syncytial virus solution;

[0018] Step 3, post-processing: the respiratory syncytial virus liquid is inactivated, hydrolyzed, and concentrated to obtain a concentrated solution; the concentrated solution is mixed with the antigen protection solution, centrifuged, frozen, and thawed and placed at 2-8°C overnight, and centrifuged and filtered again to obtain the respiratory syncytial virus antigen.

[0019] In some specific embodiments of the present invention, the antigen protection solution includes one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Tween 20, Proclin 300, sucrose or bovine serum albumin.

[0020] In some specific embodiments of the present invention, the host cell comprises a Hep2 cell.

[0021] In some specific embodiments of the present invention, the density of the adherent cells in step 1 is 5-6×10^5 cells / ml.

[0022] In some specific embodiments of the present invention, the negative number of respiratory syncytial virus infection in step 2 is 0.1.

[0023] In some specific embodiments of the present invention, the adherent host cells in step 2 are cultured for 48 to 72 hours.

[0024] In some specific embodiments of the present invention, the inactivation conditions in step 3 include inactivation using β-propiolactone. The volume ratio of the β-propiolactone to the respiratory syncytial virus solution is 1:2000. The inactivation temperature is 2-8°C; and the inactivation time is 24 hours.

[0025] In some specific embodiments of the present invention, the hydrolysis time in step 3 is 2 h.

[0026] In some specific embodiments of the present invention, the concentration factor in step 3 is 20 times.

[0027] In some specific embodiments of the present invention, the volume ratio of the concentrated solution and the antigen protection solution in step 3 is 1:1.

[0028] In some specific embodiments of the present invention, the centrifugation conditions in step 3 include centrifugation at 2000 rpm for 10 min.

[0029] In some specific embodiments of the present invention, the freezing conditions in step 3 include freezing at -20°C for 2 days.

[0030] In some specific embodiments of the present invention, the conditions for the re-centrifugation in step 3 include centrifugation at 7400 g for 10 min.

[0031] In some specific embodiments of the present invention, the pore size of the filtration in step 3 is 0.22 μm.

[0032] The invention also provides a respiratory syncytial virus antigen prepared by the preparation method.

[0033] The present invention also provides the use of the respiratory syncytial virus antigen in any of the following items:

[0034] (I) preparing a respiratory syncytial virus vaccine; and / or

[0035] (II) Prepare reagents and / or kits for detecting anti-RSV-specific IgM antibodies.

[0036] In some specific embodiments of the present invention, the respiratory syncytial virus vaccine includes an inactivated respiratory syncytial virus vaccine or a live attenuated respiratory syncytial virus vaccine.

[0037] The present invention also provides a respiratory syncytial virus vaccine, comprising the respiratory syncytial virus antigen.

[0038] The present invention also provides a reagent and / or a kit, comprising the respiratory syncytial virus antigen.

[0039] The present invention provides the following beneficial effects:

[0040] The present invention provides a method for culturing respiratory syncytial virus. The antigen prepared by the culturing method shows high activity in the antigen protection solution in the embodiment, is suitable for large-scale production, and lays a foundation for serological specific antibody detection. DETAILED DESCRIPTION

[0041] The present invention discloses a respiratory syncytial virus culture medium, a respiratory syncytial virus antigen, and a preparation method and application thereof. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0042] The invention discloses a method for efficiently culturing respiratory syncytial virus and a method for preparing and applying its natural antigen. The method for efficiently culturing respiratory syncytial virus comprises the following steps: S1, medium preparation: configuring the medium according to the reagent requirements; S2, respiratory syncytial virus culture: using the medium to culture host cells, adding respiratory syncytial virus to the host cells, 1:4 virus-carrying passage, and harvesting the virus 48-72 hours after culture; S3, respiratory syncytial virus antigen treatment and detection: inactivating the respiratory syncytial virus using β-propiolactone, adding antigen protection liquid after concentration, centrifuging and standing overnight to obtain virus liquid; diluting the virus liquid and using a respiratory syncytial IgM antibody detection kit for detection. The respiratory syncytial virus antigen prepared by the culture method has high activity, is suitable for large-scale production, and lays a foundation for serological specific antibody detection.

[0043] Unless otherwise specified, the respiratory syncytial virus culture medium, respiratory syncytial virus antigen, and raw materials and reagents used in the preparation method and application thereof provided by the present invention can all be purchased from the market.

[0044] The present invention will be further described below in conjunction with embodiments:

[0045] Preparation Example 1 Preparation of respiratory syncytial virus culture medium

[0046] The preparation process of the respiratory syncytial virus culture medium comprises the following steps:

[0047] DMEM medium solution preparation: weigh 13.4 g DMEM (gibco), 4.92 g Hepes (Sigma), 2.1 g NaHCO 3 (Sigma), 0.146 g glutamine (Sigma), 0.11 g sodium pyruvate (Sigma), 4 μL β-mercaptoethanol (Sigma), 100 KU penicillin (Biyuntian), and 0.1 g streptomycin (Biyuntian) were dissolved in 1000 mL purified water and filtered through a 0.22 μm filter.

[0048] Preparation of respiratory syncytial virus culture medium solution: Newborn calf serum (Lanzhou Rongye) was added to DMEM culture medium solution at 5% volume to obtain respiratory syncytial virus culture medium.

[0049] Preparation Example 2 Preparation of respiratory syncytial virus culture medium

[0050] The preparation process of the respiratory syncytial virus culture medium comprises the following steps:

[0051] DMEM medium solution preparation: weigh 12 g DMEM (gibco), 4 g Hepes (Sigma), 3 g NaHCO 3 (Sigma), 0.2 g glutamine (Sigma), 0.1 g sodium pyruvate (Sigma), 2 μL β-mercaptoethanol (Sigma), 100 KU penicillin (Biyuntian), and 0.1 g streptomycin (Biyuntian) were dissolved in 1000 mL purified water and filtered through a 0.22 μm filter.

[0052] Preparation of respiratory syncytial virus culture medium solution: Newborn calf serum (Lanzhou Rongye) was added to DMEM culture medium solution at 5% volume to obtain respiratory syncytial virus culture medium.

[0053] Preparation Example 3 Preparation of respiratory syncytial virus culture medium

[0054] The preparation process of the respiratory syncytial virus culture medium comprises the following steps:

[0055] DMEM medium solution preparation: weigh 14 g DMEM (gibco), 6 g Hepes (Sigma), 2 g NaHCO 3(Sigma), 0.1 g glutamine (Sigma), 0.2 g sodium pyruvate (Sigma), 8 μL β-mercaptoethanol (Sigma), 100 KU penicillin (Biyuntian), and 0.1 g streptomycin (Biyuntian) were dissolved in 1000 mL purified water and filtered through a 0.22 μm filter.

[0056] Preparation of respiratory syncytial virus culture medium solution: Newborn calf serum (Lanzhou Rongye) was added to DMEM culture medium solution at 5% volume to obtain respiratory syncytial virus culture medium.

[0057] Comparative Example 1 Preparation of respiratory syncytial virus culture medium

[0058] The preparation process of the respiratory syncytial virus culture medium comprises the following steps:

[0059] DMEM medium solution preparation: weigh 13.4 g DMEM (Gibco), 2.1 g NaHCO 3 (Sigma), 0.2 g sodium pyruvate (Sigma), 4 μL β-mercaptoethanol (Sigma), 100 KU penicillin (Biyuntian), and 0.1 g streptomycin (Biyuntian) were dissolved in 1000 mL purified water and filtered through a 0.22 μm filter.

[0060] Preparation of respiratory syncytial virus culture medium solution: Newborn calf serum (Lanzhou Rongye) was added to DMEM culture medium solution at 5% volume to obtain respiratory syncytial virus culture medium.

[0061] Comparative Example 2 Preparation of respiratory syncytial virus culture medium

[0062] The preparation process of the respiratory syncytial virus culture medium comprises the following steps:

[0063] Preparation of DMEM culture medium solution: weigh 13.4 g DMEM (gibco), 6 g Hepes (Sigma), 0.2 g sodium pyruvate (Sigma), 0.146 g glutamine (Sigma), 4 μL β-mercaptoethanol (Sigma), 100 KU penicillin (Biyuntian), and 0.1 g streptomycin (Biyuntian), dissolve in 1000 mL purified water, and filter with a 0.22 μm filter.

[0064] Preparation of respiratory syncytial virus culture medium solution: Newborn calf serum (Lanzhou Rongye) was added to DMEM culture medium solution at 5% volume to obtain respiratory syncytial virus culture medium.

[0065] Example 1 Cultivation of respiratory syncytial virus

[0066] This embodiment provides a method for preparing respiratory syncytial virus, which comprises the following steps: The respiratory syncytial virus culture medium solution used in this embodiment is obtained from Preparation Examples 1 to 5.

[0067] 1. Preparation of host cells: Hep2 cells (purchased from BMCR 1101HUM-PUMC000068) were revived in T25 cell flasks filled with respiratory syncytial virus culture medium prepared in Preparation Examples 1 to 5, respectively. After culturing at 37°C for 24 h, the old culture medium was discarded and replaced with new respiratory syncytial virus culture medium. The culture was continued at 37°C for 2 to 3 days to obtain Hep2 cells that grew a monolayer. The Hep2 cells that grew a monolayer were subcultured in a T75 cell flask at a ratio of 1:3 using the above respiratory syncytial virus culture medium, and the cells were subcultured once every 3 to 4 days. The Hep2 cells were expanded and cultured to a 10-layer cell factory according to the above subculture method, and were used for respiratory syncytial virus inoculation 4 days after subculturing to a 10-layer cell factory.

[0068] 2. Preparation of respiratory syncytial virus: Take 1 mL of respiratory syncytial virus (purchased from ATCC VR-26) and inoculate it into Hep2 cells grown in T25 square flasks. After culturing at 37°C for 2 h, discard the old culture medium and replace it with new respiratory syncytial virus culture medium. Continue to culture at 37°C for 5-7 days. When the cell lesion rate is 70-80%, freeze and thaw three times (freeze at ≤-80°C and thaw at 20-25°C) to obtain the first generation of respiratory syncytial virus. The respiratory syncytial virus inoculation is carried out at an MOI of 0.1. According to the above culture method, the respiratory syncytial virus is expanded to a 5-layer cell factory to obtain respiratory syncytial virus liquid.

[0069] 3. Virus inoculation: discard the cell supernatant of the 10-layer cell factory described in 1, and wash the Hep2 cells in the 10-layer cell factory with respiratory syncytial virus medium for 3 times; after washing, mix 1500 mL of the respiratory syncytial virus medium prepared in Preparation Example and 150 mL of the respiratory syncytial virus solution obtained in 2 (MOI is 0.1), and inoculate the mixed solution into the 10-layer cell factory. After 24 hours, carry out virus-carrying passage to the 40-layer cell factory at a ratio of 1:4. After 36 hours of virus-carrying passage, change the medium to the DMEM medium solution described in the preparation example, and culture at 37°C for 48 hours to obtain respiratory syncytial virus solution.

[0070] Example 2 Respiratory Syncytial Virus Antigen Treatment

[0071] Preparation of antigen protection solution: weigh 962.483 g purified water, 0.199 g sodium dihydrogen phosphate (Beijing Yili), 3.062 g sodium dihydrogen phosphate (Beijing Yili), 8.627 g sodium chloride (Beijing Yili), 1.032 g Tween 20 (Beijing Yili), 1.032 g Proclin300 (Sigma), 14.739 g sucrose (Beijing Yili), and 9.826 g bovine serum albumin (Sigma), and stir until completely dissolved.

[0072] Respiratory syncytial virus antigen treatment: The respiratory syncytial virus solution prepared in Example 1 was left to stand overnight at 2-8°C for precooling, and β-propiolactone was added at a volume of 1:2000 to inactivate it. It was inactivated at 2-8°C for 24 h, and hydrolyzed at 37°C for 2 h. After hydrolysis, a hemodialysis column (Shanghai Peni, 1.6 cm 2 ) was concentrated 20 times, antigen protection solution was added at a ratio of 1:1 (v:v), centrifuged at 2000 rpm for 10 min, frozen at -20℃ for 2 d, thawed at 20-25℃ and placed at 2-8℃ for 16-18 h, centrifuged at 7400 g for 10 min, filtered through a 0.22 μm filter and tested for antigen reactivity, and the remaining antigen was stored at -20℃.

[0073] Comparative Example 3 Respiratory Syncytial Virus Antigen Treatment

[0074] Preparation of antigen protection solution: weigh 962.483 g purified water, 0.199 g sodium dihydrogen phosphate (Beijing Yili), 3.062 g sodium dihydrogen phosphate (Beijing Yili), 8.627 g sodium chloride (Beijing Yili), 1.032 g Tween 20 (Beijing Yili), 1.032 g Proclin300 (Sigma), 14.739 g sucrose (Beijing Yili), and 9.826 g bovine serum albumin (Sigma), and stir until completely dissolved.

[0075] Respiratory syncytial virus antigen treatment: The respiratory syncytial virus solution prepared in Examples 1 to 3, Comparative Examples 1 or 2 was left to stand overnight at 2 to 8°C for precooling, and β-propiolactone was added at a volume of 1:2000 to inactivate it, inactivated at 2 to 8°C for 24 h, and hydrolyzed at 37°C for 2 h. After hydrolysis, a hemodialysis column (Shanghai Peni, 1.6 cm 2 ) was concentrated 20 times, antigen protection solution was added at a ratio of 1:1 (v:v), centrifuged at 2000 rpm for 10 min, frozen at -20°C for 2 d, thawed at 20-25°C, centrifuged at 7400 g for 10 min, filtered through a 0.22 μm filter, and the remaining antigen was stored at -20°C.

[0076] RSV antigen test

[0077] Respiratory syncytial virus antigen detection: The respiratory syncytial virus antigen prepared in Example 2 was diluted 1 / 15 (v / v) with antigen protection solution, and the antigen in the respiratory syncytial virus IgM antibody detection kit was used as a control. The respiratory syncytial virus IgM antibody detection kit (Antu Biological) was used for detection, and the activity of the antigen in the kit and the respiratory syncytial virus antigen prepared by the present invention were compared. The test results are shown in Table 1.

[0078] Positive samples: serum from patients infected with RSV;

[0079] Negative samples: serum from normal people;

[0080] Table 1 Antigen activity detection

[0081]

[0082] As can be seen from Table 1, the activity of the respiratory syncytial virus antigen prepared by the present invention is higher than that of the antigen in the control kit 1. After being cultured in the respiratory syncytial virus culture medium prepared in Comparative Example 1 or 2, the antigen activity after treatment is low (Control Groups 2 and 3). The respiratory syncytial virus culture medium prepared in Examples 1 to 3 of the present invention can provide the necessary growth factors, hormones, cell attachment factors and nutrients for the in vitro culture of respiratory syncytial virus, and the antigen protection solution can increase the activity of the harvested respiratory syncytial antigen, which is suitable for large-scale production. In addition, the reactivity of the antigen prepared in Example 2 of the present invention is higher than that of the antigen prepared in Comparative Example 3. It may be that under the condition of being placed at 2 to 8°C for 16 to 18 hours, Tween 20 in the antigen protection solution causes the conformation of some antigens to change, thereby improving the antigen reactivity.

[0083] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A respiratory syncytial virus culture medium, characterized in that Each liter of culture medium contains the following components: 12~14g DMEM medium, 4~6 g Hepes, 2~3g NaHCO3, 0.1~0.2 g glutamine, 0.1~0.2 g sodium pyruvate, 2~8 μL β-mercaptoethanol, 100 KU penicillin, 0.1 g streptomycin, and the balance is water.

2. The culture medium according to claim 1, characterized in that Also included is 5% by volume newborn calf serum.

3. Use of the respiratory syncytial virus culture medium as claimed in claim 1 or 2 in any of the following items: (I) culture of respiratory syncytial virus; and / or (II) Preparation of respiratory syncytial virus antigen.

4. A method for culturing respiratory syncytial virus, characterized in that: The method comprises culturing respiratory syncytial virus using the respiratory syncytial virus culture medium as claimed in claim 1 or 2.

5. A method for preparing a respiratory syncytial virus antigen, characterized in that: The steps include: Step 1, cell preparation: culturing host cells to adhere to the wall to obtain adherent host cells; Step 2, virus inoculation: mixing the respiratory syncytial virus and the respiratory syncytial virus culture medium according to claim 1 or 2, and inoculating the mixture into the adherent host cells for culture to obtain a respiratory syncytial virus liquid; Step 3, post-processing: the respiratory syncytial virus liquid is inactivated, hydrolyzed, and concentrated to obtain a concentrated solution; the concentrated solution is mixed with the antigen protection solution, centrifuged, frozen, and thawed and placed at 2-8°C overnight, and centrifuged and filtered again to obtain the respiratory syncytial virus antigen.

6. Respiratory syncytial virus antigen prepared by the preparation method as claimed in claim 5.

7. Use of the respiratory syncytial virus antigen according to claim 6 in any of the following items: (I) preparing a respiratory syncytial virus vaccine; and / or (II) Preparation of reagents and / or kits for detecting anti-RSV-specific IgM antibodies.

8. The use according to claim 7, characterized in that The respiratory syncytial virus vaccine includes an inactivated respiratory syncytial virus vaccine or a live attenuated respiratory syncytial virus vaccine.

9. A respiratory syncytial virus vaccine, characterized in that: Comprising the respiratory syncytial virus antigen as described in claim 6.

10. A reagent and / or a kit, characterized in that: Comprising the respiratory syncytial virus antigen as described in claim 6.

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