CN114410592A - Rotavirus culture medium and application thereof - Google Patents
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- CN114410592A CN114410592A CN202111648016.XA CN202111648016A CN114410592A CN 114410592 A CN114410592 A CN 114410592A CN 202111648016 A CN202111648016 A CN 202111648016A CN 114410592 A CN114410592 A CN 114410592A
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- WHGYBXFWUBPSRW-FOUAGVGXSA-N beta-cyclodextrin Chemical compound OC[C@H]([C@H]([C@@H]([C@H]1O)O)O[C@H]2O[C@@H]([C@@H](O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O[C@H]3O[C@H](CO)[C@H]([C@@H]([C@H]3O)O)O3)[C@H](O)[C@H]2O)CO)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H]3O[C@@H]1CO WHGYBXFWUBPSRW-FOUAGVGXSA-N 0.000 claims abstract description 11
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5252—Virus inactivated (killed)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/12011—Reoviridae
- C12N2720/12311—Rotavirus, e.g. rotavirus A
- C12N2720/12351—Methods of production or purification of viral material
Abstract
The invention relates to a rotavirus culture medium and application thereof, belonging to the technical field of biology. The invention provides a rotavirus culture medium, which is a DMEM culture medium added with a serum microcarrier, wherein the raw materials of the serum microcarrier comprise polyvinylpyrrolidone, beta-cyclodextrin, serum and water: the rotavirus culture medium can provide necessary growth factors, hormones, cell adhesion factors and nutrient components for the in vitro culture of Vero cells, can effectively avoid the rotavirus from being insufficiently activated due to the neutralization of pancreatin by serum, and has extremely high application prospect in the aspect of improving the titer of the harvested rotavirus liquid and further improving the quality of rotavirus vaccines.
Description
Technical Field
The invention relates to a rotavirus culture medium and application thereof, belonging to the technical field of biology.
Background
Rotavirus is one of the main pathogens causing infantile diarrhea, and mainly infects epithelial cells of small intestine to cause cell damage and cause diarrhea. The rotavirus is epidemic in summer, autumn and winter every year, the infection route is a feces-oral route, the clinical manifestation is acute gastroenteritis and permeable diarrhea, the course of the disease is generally 6-7 days, the fever lasts for 1-2 days, the vomiting lasts for 2-3 days, the diarrhea lasts for 5 days, and the dehydration symptom is seriously appeared.
Rotavirus (RV), a double-stranded ribonucleic acid virus, belongs to the family reoviridae, and is the single leading cause of diarrhea in infants and young children, almost every child of about five years of age in the world having been infected with Rotavirus at least once. There are seven rotaviruses, numbered A, B, C, D, E, F and G, of which A is the most common one, and more than 90% of cases of human rotavirus infection are caused by the same.
In China, about 1000 million infants suffer from rotavirus infectious gastroenteritis every year, which accounts for 1/4 in infants, and is the most main pathogen causing severe diarrhea of infants. Rotavirus is firstly discovered in 1973, but no specific medicine exists until now, and the antibiotic treatment has no obvious effect. At this stage, vaccination with vaccines remains the only effective means of prevention and control of rotavirus infection.
At present, the cell matrix used for producing human rotavirus vaccine is mainly Vero cells. Vero cells are a vaccine production cell line approved by the world health organization. Compared with primary cells, diploid cells and other passage cell matrixes used for vaccine production, the Vero cells have the advantages of continuous passage, high growth speed, stable genetic character, low malignant transformation degree, high biological safety, low requirement on culture conditions, easiness in large-scale culture and the like.
As adherent fibroblasts, Vero cells are serum-dependent, requiring serum to provide essential growth factors, hormones, cell adhesion factors and nutrients for their in vitro culture. Serum used as a supplement component of a culture medium for Vero cell culture and vaccine preparation has many defects, for example, serum has a certain inhibiting effect on rotavirus and needs to be activated by pancreatin to well infect viruses of cells, but the serum can neutralize the pancreatin to ensure that the rotavirus can not be fully activated, so that the titer of the harvested rotavirus solution is reduced, and the quality of the rotavirus vaccine is further influenced.
Disclosure of Invention
In order to solve the problems, the invention provides a rotavirus culture medium, which is a DMEM culture medium added with a serum microcarrier; the raw materials of the serum microcarrier comprise polyvinylpyrrolidone, beta-cyclodextrin, serum and water.
In one embodiment of the invention, the serum microcarrier is added in an amount of 15-30 g/L in a DMEM medium.
In one embodiment of the present invention, in the serum microcarrier, the mass ratio of the polyvinylpyrrolidone, the beta-cyclodextrin and the serum is 0.5-2: 1: 0.1.
in one embodiment of the present invention, in the serum microcarrier, the mass ratio of the polyvinylpyrrolidone, the β -cyclodextrin and the serum is 1: 1: 0.1.
the invention provides application of the rotavirus culture medium in preparation of rotavirus vaccines.
In one embodiment of the invention, the rotavirus vaccine is a rotavirus inactivated vaccine or a rotavirus attenuated live vaccine.
The invention provides a method for preparing rotavirus inactivated vaccine, which comprises the following steps:
cell preparation: culturing the host cell to be adherent to obtain an adherent host cell;
virus inoculation: mixing rotavirus, the rotavirus culture medium and pancreatin, and inoculating the mixture to adherent host cells for culture to obtain rotavirus liquid;
and (3) post-treatment: and crushing the rotavirus solution, and then sequentially performing centrifugation, filtration, inactivation, concentration and purification to obtain the rotavirus inactivated vaccine.
The invention provides application of the method in preparation of rotavirus inactivated vaccine.
The invention provides a method for preparing rotavirus attenuated live vaccine, which comprises the following steps:
cell preparation: culturing the host cell to be adherent to obtain an adherent host cell;
virus inoculation: mixing a rotavirus attenuated strain, the rotavirus culture medium and pancreatin, and inoculating the mixture to adherent host cells for culture to obtain a rotavirus liquid;
and (3) post-treatment: and crushing the rotavirus solution, and then sequentially carrying out centrifugation, filtration and concentration to obtain the rotavirus inactivated vaccine.
The invention provides application of the method in preparing attenuated live rotavirus vaccine.
The technical scheme of the invention has the following advantages:
the invention provides a rotavirus culture medium, which is a DMEM culture medium added with a serum microcarrier, wherein the raw materials of the serum microcarrier comprise polyvinylpyrrolidone, beta-cyclodextrin, serum and water: the rotavirus culture medium can provide necessary growth factors, hormones, cell adhesion factors and nutrient components for the in vitro culture of Vero cells, can effectively avoid the rotavirus from being insufficiently activated due to the neutralization of pancreatin by serum, and has extremely high application prospect in the aspect of improving the titer of the harvested rotavirus liquid and further improving the quality of rotavirus vaccines.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The following examples do not show specific experimental procedures or conditions, and can be performed according to the procedures or conditions of the conventional experimental procedures described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Example 1: rotavirus culture medium
The embodiment provides a rotavirus culture medium, wherein the rotavirus culture medium is a DMEM culture medium added with a serum microcarrier; the raw materials of the serum microcarrier comprise polyvinylpyrrolidone, beta-cyclodextrin, fetal calf serum and water; in the serum microcarrier, the mass ratio of polyvinylpyrrolidone, beta-cyclodextrin and fetal calf serum is 1: 1: 0.1.
the preparation process of the rotavirus culture medium comprises the following steps:
preparation of microcarrier solution: weighing 100mg of polyvinylpyrrolidone (K60, purchased from Shanghai Aladdin Biotechnology, Ltd.), 100mg of beta-cyclodextrin (purchased from Shanghai Aladdin Biotechnology, Ltd.) and 10mg of fetal bovine serum (purchased from Sigma) and dissolving in 5mL of deionized water, ultrasonically mixing for 20min at 25 ℃ and 30HKz, stirring for 18h at 25 ℃ and 150rpm, and concentrating in a 65 ℃ oven for 12h to obtain a microcarrier solution;
preparation of serum microcarriers: adding the microcarrier solution into a spherical mold, vacuumizing for 15min in a vacuum pump at 25 ℃, taking out the spherical mold from the vacuum pump, placing the spherical mold in a 35 ℃ oven for drying for 12h, and demolding to obtain a spherical serum microcarrier with the diameter of 1 mm;
preparation of rotavirus culture medium: serum microcarriers were added to DMEM medium (purchased from Gibco) at an addition level of 25g/L to give a rotavirus medium.
Example 2: method for preparing rotavirus liquid
This example provides a method of preparing rotavirus solution, comprising the steps of:
preparation of host cells: recovering Vero cells (the Vero cells are CCL-81 purchased from ATCC) to a T25 cell bottle filled with rotavirus culture medium (the rotavirus culture medium is DMEM culture medium purchased from Gibco company), culturing at 37 ℃ for 24h, discarding the old culture medium, replacing new virus culture medium, and continuously culturing at 37 ℃ for 7d to obtain Vero cells with full monolayers; subculturing full monolayer Vero cells in a T75 cell bottle according to the ratio of 1:6 by using the virus culture medium, and subculturing for 1 time after 7 d; the Vero cells are expanded and cultured to a 10-layer cell factory according to the subculture method, and are used for rotavirus inoculation after being subcultured to a 10-layer cell factory for 4 d;
activation of rotavirus: taking 1mL of rotavirus (rotavirus is SA11 from American ATCC), adding pancreatin (from Gibco company) with a final concentration of 20 μ g/mL and calcium chloride (from Sigma company) with a final concentration of 500 μ g/mL into the virus solution, and activating in a water bath at 37 ℃ for 1 h;
virus inoculation: discarding the cell supernatant of the cell factory, washing Vero cells in 10-layer cell factory with rotavirus culture medium (here, the rotavirus culture medium is DMEM culture medium purchased from Gibco company) for 3 times to remove serum; after the cleaning, 2L of the rotavirus culture medium prepared in the embodiment 1 is mixed with 150 μ L of the activated rotavirus liquid to obtain a mixed liquid; pancreatin (purchased from Gibco) was added to the mixture to a final concentration of 20. mu.g/mL to obtain a seed inoculum; the inoculum was inoculated at 200 mL/layer into a 10-layer cell factory and cultured at 37 ℃ for 3 days to obtain rotavirus solution. The operation is repeated three times to obtain three batches of rotavirus liquid which are respectively named as #1, #2 and # 3.
Comparative example 1: method for preparing rotavirus liquid
This comparative example provides a method of preparing rotavirus solution, which method comprises: on the basis of example 2, the rotavirus culture medium prepared in example 1 was replaced with a DMEM medium to obtain a rotavirus solution. The operation is repeated three times to obtain three batches of rotavirus liquid which are respectively named as #1, #2 and # 3.
Comparative example 2: method for preparing rotavirus liquid
This comparative example provides a method of preparing rotavirus solution, which method comprises: in addition to example 2, the rotavirus culture medium prepared in example 1 was replaced with DMEM medium supplemented with 15% (v/v) fetal bovine serum to obtain a rotavirus solution. The operation is repeated three times to obtain three batches of rotavirus liquid which are respectively named as #1, #2 and # 3.
Experimental example 1: effect of rotavirus Medium on Virus titer of rotavirus fluid
The experimental example provides an experiment for the influence of a rotavirus culture medium on the virus titer of rotavirus liquid, and the experimental process is as follows:
the results of measuring infectious rotavirus titers in the rotavirus solutions prepared in example 2 and comparative examples 1 to 2 by using a cytopathic method (pharmacopoeia section three) are shown in table 1.
As can be seen from table 1, the rotavirus culture medium prepared in example 1 can provide necessary growth factors, hormones, cell adhesion factors and nutritional ingredients for in vitro culture of Vero cells, and can effectively avoid insufficient activation of rotavirus caused by serum neutralization of pancreatin, thereby having a very high application prospect in increasing the titer of the harvested rotavirus liquid and further improving the quality of rotavirus vaccines.
TABLE 1 Effect of rotavirus culture Medium on rotavirus broth virus titer
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A rotavirus culture medium is characterized in that the rotavirus culture medium is a DMEM culture medium added with a serum microcarrier; the raw materials of the serum microcarrier comprise polyvinylpyrrolidone, beta-cyclodextrin, serum and water.
2. The rotavirus culture medium of claim 1, wherein the serum microcarrier is added in an amount of 15-30 g/L in DMEM medium.
3. The rotavirus culture medium of claim 1 or 2, wherein in the serum microcarrier, the mass ratio of the polyvinylpyrrolidone, the beta-cyclodextrin and the serum is 0.5-2: 1: 0.1.
4. the rotavirus culture medium of any one of claims 1 to 3, wherein in the serum microcarrier, the mass ratio of polyvinylpyrrolidone, beta-cyclodextrin and serum is 1: 1: 0.1.
5. use of a rotavirus culture medium of any one of claims 1 to 4 in the preparation of a rotavirus vaccine.
6. The use of claim 5, wherein the rotavirus vaccine is a inactivated rotavirus vaccine or a live attenuated rotavirus vaccine.
7. A method for preparing inactivated rotavirus vaccine, which comprises the following steps:
cell preparation: culturing the host cell to be adherent to obtain an adherent host cell;
virus inoculation: mixing rotavirus, the rotavirus culture medium of any one of claims 1 to 4 and pancreatin, and then inoculating the mixture into adherent host cells for culture to obtain a rotavirus solution;
and (3) post-treatment: and crushing the rotavirus solution, and then sequentially performing centrifugation, filtration, inactivation, concentration and purification to obtain the rotavirus inactivated vaccine.
8. Use of the method of claim 7 for the preparation of inactivated rotavirus vaccine.
9. A method for preparing a live attenuated rotavirus vaccine, comprising the steps of:
cell preparation: culturing the host cell to be adherent to obtain an adherent host cell;
virus inoculation: mixing a rotavirus attenuated strain, the rotavirus culture medium as claimed in any one of claims 1 to 4 and pancreatin, and inoculating the mixture to adherent host cells for culture to obtain a rotavirus liquid;
and (3) post-treatment: and crushing the rotavirus solution, and then sequentially carrying out centrifugation, filtration and concentration to obtain the rotavirus inactivated vaccine.
10. Use of the method of claim 9 for the preparation of live attenuated rotavirus vaccine.
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