Way to get the rubella virus and supportive nutrient medium for cultivation (options)

 

(57) Abstract:

The invention relates to receiving vaccine and diagnostic products rubella virus. The method involves infecting diploid cell cultures by the rubella virus and subsequent cultivation of the virus in supporting nutrient medium. As a diploid cell cultures using lung fibroblasts human embryo FLACH 385/13 (basic branch of the Russian collection of cells at the research Institute of influenza, Russian Academy of medical Sciences, ALAC(DP)004). Supporting nutrient medium contains medium 199, medium Needle with a double amino acid content. According to the first variant it added aminopeptide and calcium chloride, and the second - arginine and calcium chloride. The invention increases the biological activity of drugs rubella virus and provides sufficient purity of the final product by eliminating allergenic factor. 3 C. p. F.-ly, 3 tables.

The invention relates to medical biotechnology and can be used when receiving vaccine and diagnostic products rubella virus.

A method of obtaining experienced a series of cultural vaccine against rubella, involving infection with rubella virus Pervy ivalsa culture medium and collecting the viral mass (Cm. C. N. Investment activity. The experience of receiving and attenuate rubella strain eagles/In kN.: Respiratory virus infection, " Proc. IEM them. Pasteur, L. - 1973, I. XLII, S. 137 - 143).

However, this method provides enough high yield of the target product from the moderate sensitivity of cell culture from kidney fortnight rabbits. Harvest rubella virus during the first ten passages ranged from 3.0 to 4.0 lg TCPD50/0,5 ml in Addition, obtaining primary trypsinization culture of kidney cells fortnight rabbits (CPD) associated with a significant amount of material resources for the purchase of animals from specialized farms with strictly regulated conditions and constant examination on the absence of a number of viral and microbial agents, and getting viral drugs on CPD, in turn, is associated with a certain complexity of making a fabric substrate.

The closest to the essence of the claimed invention is a method of obtaining a culture vaccine against rubella, involving infection with rubella virus diploid cell culture W1-38, received the Hayflick from the lungs of human embryos, followed PMC in human fibroblasts: evidence for reduced nasopharyngeal extention. - Amer. J. Epidem., 1967, - 86, - p. 468 - 477, ). However, the harvest of rubella virus (units RA 27/3) did not exceed 4.5 lg TCPD50/0,5 ml in Addition, enrolling in Russia foreign cell lines, promising for the production of viral preparations, very limited, and the creation of the gene pool of these cells, providing commercial production, is almost impossible. Despite the large number of new diploid lines and strains in our country is not such that would be recommended as a substrate to obtain a culture rubella vaccines and diagnostic products and could provide their commercial production.

Known supporting nutrient medium for cultivation of rubella virus - medium 199 (a method of obtaining a vaccine against rubella. Patent N 4147772 USA. Appl. 29.07.67, Publ. 03.04.79). However, it does not ensure receipt of rubella virus to high titer infectious activity.

The closest entity to the claimed nutrient medium is medium 199 supplemented with 5% aminopeptide or 5% inactivated calf serum (Cm. Investment Activity C. N. "The experience of receiving and attenuate rubella strain eagles/In kN.: Respiratory virus infection, " Proc. IEM them. Pasteur. - 1973.50/0,5 ml Add 5% inactivated calf serum does not provide a sufficient purity of the final product, because of its presence in the preparation of a vaccine is a potential allergenic factor for individuals with a heightened sensitivity to alien squirrel. The presence of serum (even trace its quantities in diagnostic preparations may serve as a factor decreasing the specificity of the reaction. In addition, the serum of animals can be contaminated by extraneous viral and microbial agents.

The invention is aimed at developing a method of producing rubella virus and supportive nutrient medium for cultivation increased biological activity of drugs rubella virus and is provided with sufficient purity of the final product by eliminating allergenic factor.

The essence of the invention is as follows.

The method of obtaining rubella virus provides for the infection diploid cell cultures by the rubella virus and subsequent cultivation of the virus in supporting nutrient medium. As a diploid cell cultures using lung fibroblasts human embryo - line 38004, authors: Litvinchuk L. F., look Etc., Shitikova, C.). Line derived from lung 17-18 weeks of normal human embryo and cryopreserved in 5th and 7th passages. Cultivated static and roller means has a fibroblast-like type of growth, limited 50 passages of life. The line of sight confirmed kariologicheskii; bacteria, fungi, Mycoplasma is not detected; carcinogenic potencies in the test using organ culture of the skin of the chicken embryo is not revealed.

Obviously the advantage of using FLACH for improving the technology of production of the vaccine compared with primary trypsinization cell cultures due to the simplicity and efficiency of the first of them, the possibility of comprehensive attestovannoi in accordance with the requirements of the world health organization in respect of oncogenic security, lack of third contamination and stability of the biological properties of the cell line. Additionally, in the case of primary trypsinization cell culture CPD final product is contaminated ballast proteins formed as a result of trypsinization renal tissue and firmly enough adsorbiruyuschimi on a surface is nuxa extremely diverse and may vary depending on the virus strain. Research carried out by the authors, showed that they used as a substrate for reproduction of rubella virus diploid line FLACH-385/13 is the most sensitive (the concentration of the virus even without prior adaptation to the cell culture reaches 4.5 to 5.0 lg TCPD50/0.5 ml) of the whole range of investigated cell cultures, such as the kidney of a human embryo (PAC), skin and muscle fibroblasts of the human embryo (FAC), as well as primary trypsinization kidney of the newborn rabbit (PL. 1).

Through the use of this diploid lines were able to increase the biological activity of drugs rubella virus.

Supporting nutrient medium for cultivation of rubella virus in the first embodiment contains medium 199 and aminopeptide. To increase the biological activity of drugs rubella virus and increase the purity of the final product is additionally introduced Wednesday Needle MEME with double aminoacids and calcium chloride in the following ratio of ingredients,%:

Environment 199 - 40.....54

Wednesday Needle MEME with dual amino acid - 40....54

Aminopeptide - 1.....5

Calcium chloride - 5.....10

Before you add in pitate to medium 199 was added Wednesday Needle MEME with double amino acid content, calcium chloride and arginine in the following ratio of ingredients,%:

Environment 199 - 40.....54

Wednesday Needle MEME with dual amino acid - 40....54

Arginine - 5.....10

Calcium chloride - 1.....5

Before adding to the culture medium arginine, was preparing its 2.5% concentrate, calcium chloride - 2% concentrate.

In the proposed compositions of culture media refused traditionally used in the composition of the nutrient medium that supports the reproduction of the virus rubella, fetal calf serum or bovine serum, because this component can serve as a factor that may cause undesired anaphylactogenic reactions in individuals with increased sensitivity to even trace amounts of protein.

To compensate for the nutrients contained in the serum and to support the reproduction of the virus at a high level, include components such as 2% CaCl2(1-10%), 2,5% arginine (5-1%) and aminopeptide (1-5%). The concentration of virus in the final product when it reaches 5.5 to 6.0 lg TCPD50/0.5 ml (table. 2 and 3), which shows not only a compensatory role of the above components Pete the SJ as follows.

Example 1. The cultivation of rubella virus in static conditions.

Mattresses with a volume of 250 ml with cell culture FLACH 385/13 thrice washed from serum phosphate-saline buffer solution, make 5 ml supporting medium, containing the estimated amount of the seed virus.

Mattresses with a volume of 250 ml with cell culture FLACH 385/13 thrice washed from serum phosphate-saline buffer solution, make 5 ml supporting medium, containing the estimated amount of the seed virus (multiplicity of infection for strain eagles-B" is 0,01 - 0,001 TCPD50/cell), conduct the adsorption of virus for 1.5 hours at room temperature, then add 35 ml of a supporting medium, consisting of

eagle medium MEM - 40%

environment 199 - 45%

aminopeptide - 5%

2% CaCl2- 10%

and 250 µg/ml streptomycin and kanamycin.

The infected cells are incubated at a temperature of 34-45oC, daily recording state of the monolayer, the development cytopathogenic steps of the virus and making charges vaccinated cultural liquid 5, 8, 11, 14 day etc. complete degradation of the cell monolayer. Viral fees cooled at 4 is received vaccinated culture liquid together with exfoliating the affected cells retain from use at -20...-40oC. Infectious titer of the virus determined by titration on human cell cultures kidney rabbit RK13 equal to 4,5 - 6,0 lg TCPD50/0,5 ml

Example 2. The cultivation of rubella virus in roller conditions.

Roller bottles with a volume of 1 or 3 liters of cell culture FLACH 385/13 triple washed from serum phosphate-saline buffer solution, make 10 or 30 ml, respectively, supporting nutrient medium containing 0,1 - 0,01 TCPD50/cell seeding rubella virus (PCs "eagles-B"), spend the adsorption of virus for 1.5 - 2 hours at 37oC, then add 90 or 270 ml, respectively, supporting the nutrient medium, consisting of

eagle medium MEM - 45%

environment 199 - 40%

2,5% arginine - 10%

2% CaCl2- 5%

and 250 µg/ml streptomycin and kanamycin.

The infected cells are incubated at a temperature of 34-35oC on a roller setup. At least development cytopathogenic steps of the virus do 3 - 4 collection vaccinated culture fluid, ranging from 50% up to a total destruction of the monolayer of cells. Viral fees three-frozen in dry ice and alcohol and thawed in a water bath at 37oC. Virussolaris, add the stabilizer and lyophilizer or store at -20...-40oC. Control of biological activity vaccinated culture fluid is performed on human cell cultures kidney rabbit RK13. The infectious titer of the virus is 5.0 to 6.0 lg TCPD50/0,5 ml

Example 3. Preparation of supporting nutrient medium

(option 1)

To get the first version supporting nutrient medium pre-prepared 2% (wt.%) the solution of calcium chloride in phosphate-saline buffer solution, sterilized it by autoclaving and stored at +4oC for 6 months. On Wednesday Needle MEME with double amino acid content add double the amount of glutamine. Mix all the ingredients in the following sequence: Wednesday Needle MEME (45%), medium 199 (40%), aminopeptide (5%) and 2% CaCl2(10%). To the mixture was added to 250 μg/ml of streptomycin and kanamycin and contribute vials with an infected monolayer cells.

Example 4. Preparation of supporting nutrient medium

(option 2)

To obtain the second option supports nutrient medium pre-prepared 2% (wt.%) the solution of calcium chloride, as in example 3. a 2.5% (wt.%) adopted at +4oC for 6 months. On Wednesday Needle MEME with double amino acid content add double the amount of glutamine. Mix all the ingredients in the following sequence: Wednesday Needle MEME (45%), medium 199 (40%), 2.5% arginine (10%) and 2% CaCl2(5%). To the mixture was added to 250 μg/ml of streptomycin and kanamycin and contribute vials with an infected monolayer cells.

Thus, the use of a diploid cell culture FLACH 385/13 and proposed structures supporting nutrient medium allows a 10 - to 100-fold increase in the yield of rubella virus-free serum components, compared with primary trypsinization cell culture kidneys 2-week-old rabbit, and other diploid cell cultures, which gives the opportunity to use the described method for obtaining biomass of rubella virus in stable, carefully tantrayana and available cellular substrate of human origin and application of acquired viral intermediate product in the manufacture of vaccine and diagnostic products.

1. The method of obtaining rubella virus, providing for the infection diploid cell cultures by rubella virus with subsequent cultivar is tury use lung fibroblasts human embryo FLACH 385/13 (basic branch of the Russian collection of cells at the research Institute of influenza, Russian Academy of medical Sciences, ALAC(DP)004).

2. Supporting nutrient medium for cultivation of rubella virus-containing medium 199 and aminopeptide, characterized in that it further comprises a Needle MEME with double aminoacids and calcium chloride in the following ratio of ingredients,%:

Wednesday 199 - 40 - 54

Wednesday Needle MEME with dual amino acid - 40 - 54

Aminopeptide - 1 - 5

Calcium chloride - 5 - 10

3. Supporting nutrient medium for cultivation of rubella virus-containing medium 199, characterized in that it further comprises a Needle MEME with double amino acid, calcium chloride and arginine in the following ratio of ingredients,%:

Wednesday 199 - 40 - 54

Wednesday Needle MEME with dual amino acid - 40 - 54

Arginine - 5 - 10

Calcium chloride - 1 - 5

 

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