阅读说明：本技术 一种用于st细胞培养及相应病毒生产的低血清培养基 (Low-serum culture medium for ST cell culture and corresponding virus production ) 是由 房圆瑗 齐智 刘海英 孟丹丹 于 2019-09-23 设计创作，主要内容包括：本发明公开了一种用于ST细胞培养及相应病毒生产的低血清培养基,包括以下组成成分：氨基酸、维生素、无机盐类、辅助成分和蛋白质；所述氨基酸包括甘氨酸、盐酸精氨、谷氨酰胺、盐酸赖氨酸、酪氨酸二钠盐二水合物；所述蛋白质包括重组胰岛素、4-羟乙基哌嗪乙磺酸(HEPES)；本发明制备的用于ST细胞培养及相应病毒生产的低血清培养基中,使用甲骨文生物ST低血清培养基IV系列添加3%猪瘟专用新生牛血清情况下能够替代传统细胞培养基对ST细胞进行常规细胞培养,且细胞培养状态与数量均与传统培养方法无显著差异,保持细胞良好的生长状态；同时利用该培养体系可以较好地增殖猪瘟病毒,病毒滴度不小于50万倍RID。(The invention discloses a low serum culture medium for ST cell culture and corresponding virus production, which comprises the following components: amino acids, vitamins, inorganic salts, auxiliary components and proteins; the amino acid comprises glycine, arginine hydrochloride, glutamine, lysine hydrochloride and tyrosine disodium salt dihydrate; the protein comprises recombinant insulin, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES); in the low serum culture medium prepared by the invention and used for ST cell culture and corresponding virus production, the conventional cell culture can be performed on ST cells by replacing the conventional cell culture medium under the condition that 3% special newborn calf serum for swine fever is added to an Ornithogen ST low serum culture medium IV series, and the cell culture state and the number have no obvious difference from those of the conventional culture method, so that the good growth state of the cells is maintained; meanwhile, the culture system can be used for better propagating the classical swine fever virus, and the virus titer is not less than 50 ten thousand times RID.)

1. A low serum medium for ST cell culture and production of corresponding viruses, comprising the following components: amino acids, vitamins, inorganic salts, auxiliary components and proteins; the amino acid comprises 5-10mg/L of glycine, 5-10mg/L of alanine, 110-150mg/L of arginine hydrochloride, 10-15mg/L of asparaginyl monohydrate, 10-15mg/L of aspartic acid, 20-40mg/L of cystine dihydrochloride, 10-15mg/L of glutamic acid, 200-350mg/L of glutamine, 25-50mg/L of histidine monohydrochloride, 25-60mg/L of isoleucine, 25-60mg/L of leucine, 50-80mg/L of lysine hydrochloride, 10-20mg/L of methionine, 20-40mg/L of phenylalanine, 10-15mg/L of proline, 10-15mg/L of serine, 25-50mg/L of threonine, 5-15mg/L tryptophan, 30-60mg/L tyrosine disodium salt dihydrate, 25-50mg/L valine and 5-10mg/L hydroxyproline; the protein comprises 5-10mg/L of human transferrin, 5-10mg/L of recombinant insulin, and 3400mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid 1500-.

2. The low serum medium according to claim 1 for ST cell culture and production of corresponding viruses, the vitamin is characterized by comprising 0.05-0.2mg/L of biotin, 2-5mg/L, D-calcium pantothenate, 2-5mg/L of folic acid, 2-5mg/L of nicotinamide, 0.1-0.3mg/L of pyridoxine hydrochloride, 0.1-0.3mg/L of pyridoxal hydrochloride, 0.1-0.3mg/L of riboflavin, 1-5mg/L, B12 of thiamine hydrochloride, 0.1-0.3mg/L, i-inositol, 5-10mg/L of vitamin A acetate, 0.01-0.05mg/L of vitamin D, and 0.005-0.01mg/L of vitamin K.

3. The low serum culture medium for ST cell culture and production of corresponding viruses of claim 2, wherein the inorganic salts comprise anhydrous calcium chloride 150-200mg/L, anhydrous magnesium sulfate 50-120mg/L, potassium chloride 300-400mg/L, sodium bicarbonate 2500-3700mg/L, sodium chloride 5000-6000mg/L, sodium dihydrogen phosphate monohydrate 120-150mg/L, copper sulfate pentahydrate 0.0005-0.001mg/L, ferrous sulfate heptahydrate 0.1-0.5mg/L, and anhydrous magnesium chloride 10-30 mg/L.

4. The low serum medium as claimed in claim 3, wherein the auxiliary components include 5000mg/L D-glucose 2000-, 1-5mg/L ethanolamine, 0.1-0.5mg/L hypoxanthine sodium, 0.1-0.5mg/L linoleic acid, 10-15mg/L phenol red, 2-6mg/L putrescine dihydrochloride, 90-120mg/L sodium pyruvate, 0.5-2.0mg/L reduced glutathione, 300mg/L galactose 100-, 5-15mg/L adenine, 5-15mg/L serotonin, 1-5mg/L water-soluble cholesterol, 10-20mg/L guanine hydrochloride, 0.1-0.5mg/L thymine, Uracil 0.1-0.5mg/L, thymidine 0.1-0.5 mg/L.

5. The low serum medium for ST cell culture and production of a corresponding virus according to claim 4, comprising the following components: 7.5mg/L glycine, 8.9mg/L alanine, 126mg/L arginine hydrochloride, 13.2mg/L asparagine monohydrate, 13.3mg/L aspartic acid, 31.28mg/L cystine dihydrochloride, 14.7mg/L glutamic acid, 292mg/L glutamine, 42mg/L histidine monohydrochloride, 52mg/L isoleucine, 52mg/L leucine, 72.5mg/L lysine hydrochloride, 15mg/L methionine, 32mg/L phenylalanine, 11.5mg/L proline, 10.5mg/L serine, 48mg/L threonine, 10mg/L tryptophan, 51.9mg/L tyrosine disodium salt dihydrate, 46mg/L valine, 6.8mg/L hydroxyproline, 0.15mg/L biotin, 3.5mg/L, D-calcium pantothenate, 3.5mg/L folic acid, 3.5mg/L nicotinamide, pyridoxine hydrochloride, 0.2mg/L pyridoxal hydrochloride, 0.1mg/L pyridoxal hydrochloride, 0.25mg/L riboflavin, thiamine hydrochloride, 3.5mg/L, B12 vitamin, 0.2mg/L, i-inositol, 6.3mg/L vitamin A acetate, 0.05mg/L vitamin D, 0.07mg/L vitamin K0.008mg/L vitamin, anhydrous calcium chloride, 180mg/L anhydrous magnesium sulfate, 380mg/L potassium chloride, 2700mg/L sodium bicarbonate, 5600mg/L sodium chloride, 130mg/L sodium dihydrogen phosphate monohydrate, 0.001mg/L copper sulfate pentahydrate, 0.3mg/L ferrous sulfate heptahydrate, 25mg/L anhydrous magnesium chloride, 4000mg/L of D-glucose, 2.5mg/L of ethanolamine, 0.4mg/L of hypoxanthine sodium, 0.2mg/L of linoleic acid, 12mg/L of phenol red, 5.5mg/L of putrescine dihydrochloride, 105mg/L of sodium pyruvate, 1.7mg/L of reduced glutathione, 200mg/L of galactose, 12.5mg/L of adenine, 5.5mg/L of serotonin, 2.8mg/L of water-soluble cholesterol, 14.5mg/L of guanine hydrochloride, 0.2mg/L of thymine, 0.2mg/L of uracil, 0.38mg/L of thymidine, 6.5mg/L of human transferrin, 10mg/L of recombinant insulin and 2800mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid.

6. The low serum medium for ST cell culture and production of the corresponding virus of claim 5, wherein the human transferrin is saturated iron human transferrin.

7. The low serum medium for ST cell culture and production of a corresponding virus according to claim 5, wherein the recombinant insulin is a full chain recombinant insulin.

Technical Field

The invention relates to the field of production of biopharmaceutical vaccines, in particular to a low serum culture medium for ST cell culture and production of corresponding viruses.

Background

The traditional culture mode of ST cell culture is that 10% calf serum is added into MEM culture medium to culture cells, the serum consumption is large, and the cost is high.

The ST cells are relatively dependent on serum in the culture process, so that the conditions of slow cell growth, cell death and the like in serum culture are reduced; the ST low serum culture medium in the market has multiple additive components and complex components on the basis of a common basic culture medium. When the hog cholera virus is produced, the cells are not firmly adhered to the wall after virus inoculation, the stable production is difficult, the virus production effect is poor, and the quality requirement of a semi-finished product is not met; based on this, we propose a low serum medium for ST cell culture and production of the corresponding virus.

Disclosure of Invention

The object of the present invention is to provide a low serum medium for ST cell culture and production of corresponding viruses, which solves the problems mentioned above in the background art.

In order to achieve the purpose, the invention provides the following technical scheme:

a low serum medium for ST cell culture and production of corresponding viruses, comprising the following components: amino acids, vitamins, inorganic salts, auxiliary components and proteins; the amino acid comprises 5-10mg/L of glycine, 5-10mg/L of alanine, 110-150mg/L of arginine hydrochloride, 10-15mg/L of asparaginyl monohydrate, 10-15mg/L of aspartic acid, 20-40mg/L of cystine dihydrochloride, 10-15mg/L of glutamic acid, 200-350mg/L of glutamine, 25-50mg/L of histidine monohydrochloride, 25-60mg/L of isoleucine, 25-60mg/L of leucine, 50-80mg/L of lysine hydrochloride, 10-20mg/L of methionine, 20-40mg/L of phenylalanine, 10-15mg/L of proline, 10-15mg/L of serine, 25-50mg/L of threonine, 5-15mg/L tryptophan, 30-60mg/L tyrosine disodium salt dihydrate, 25-50mg/L valine and 5-10mg/L hydroxyproline; the protein comprises 5-10mg/L of human transferrin, 5-10mg/L of recombinant insulin, and 3400mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) 1500-.

As a further scheme of the invention: the vitamins include biotin 0.05-0.2mg/L and choline chloride

2-5mg/L, D-calcium pantothenate 2-5mg/L, folic acid 2-5mg/L, nicotinamide 2-5mg/L, pyridoxine hydrochloride 0.1-0.3mg/L, pyridoxal hydrochloride 0.1-0.3mg/L, riboflavin 0.1-0.3mg/L, thiamine hydrochloride 1-5mg/L, B12 vitamin 0.1-0.3mg/L, i-inositol 5-10mg/L, vitamin A acetate 0.01-0.05mg/L, vitamin D

0.01-0.1mg/L and 0.005-0.01mg/L of vitamin K.

As a still further scheme of the invention: the inorganic salts comprise 150-200mg/L of anhydrous calcium chloride, 50-120mg/L of anhydrous magnesium sulfate, 300-400mg/L of potassium chloride, 2500-3700mg/L of sodium bicarbonate, 5000-6000mg/L of sodium chloride, 120-150mg/L of monobasic sodium phosphate, 0.0005-0.001mg/L of blue vitriol, 0.1-0.5mg/L of ferrous sulfate heptahydrate and 10-30mg/L of anhydrous magnesium chloride.

As a still further scheme of the invention: the auxiliary components comprise 5000mg/L of D-glucose 2000-containing-ketone, 1-5mg/L of ethanolamine, 0.1-0.5mg/L of hypoxanthine sodium, 0.1-0.5mg/L of linoleic acid, 10-15mg/L of phenol red, 2-6mg/L of putrescine dihydrochloride, 90-120mg/L of sodium pyruvate, 0.5-2.0mg/L of glutathione, 300mg/L of galactose-containing-ketone, 5-15mg/L of adenine, 5-15mg/L of serotonin, 1-5mg/L of water-soluble cholesterol, 10-20mg/L of guanine hydrochloride, 0.1-0.5mg/L of thymine, 0.1-0.5mg/L of uracil and 0.1-0.5mg/L of thymidine.

As a still further scheme of the invention: comprises the following components: 7.5mg/L glycine, 8.9mg/L alanine, 126mg/L arginine hydrochloride, 13.2mg/L asparagine monohydrate, 13.3mg/L aspartic acid, 31.28mg/L cystine dihydrochloride, 14.7mg/L glutamic acid, 292mg/L glutamine, 42mg/L histidine monohydrochloride, 52mg/L isoleucine, 52mg/L leucine, 72.5mg/L lysine hydrochloride, 15mg/L methionine, 32mg/L phenylalanine, 11.5mg/L proline, 10.5mg/L serine, 48mg/L threonine, 10mg/L tryptophan, 51.9mg/L tyrosine disodium salt dihydrate, 46mg/L valine, 6.8mg/L hydroxyproline, 0.15mg/L biotin, 3.5mg/L choline chloride, 3.5mg/L, 3.5mg/L of D-calcium pantothenate, 3.5mg/L of folic acid, 3.5mg/L of nicotinamide, 0.2mg/L of pyridoxine hydrochloride, 0.1mg/L of pyridoxal hydrochloride, 0.25mg/L of riboflavin, 3.5mg/L, B12 of thiamine hydrochloride, 0.2mg/L, i of vitamin, 6.3mg/L of inositol, 0.05mg/L of vitamin A acetate, D0.07mg/L of vitamin, 0.008mg/L of vitamin K, 180mg/L of anhydrous calcium chloride, 92mg/L of anhydrous magnesium sulfate, 380mg/L of potassium chloride, 2700mg/L of sodium bicarbonate, 5600mg/L of sodium chloride, 130mg/L of monobasic sodium phosphate, 0.001mg/L of copper sulfate pentahydrate, 0.3mg/L of ferrous sulfate heptahydrate, 25mg/L, D of anhydrous magnesium chloride, 4000mg/L of glucose, Ethanolamine 2.5mg/L, hypoxanthine sodium 0.4mg/L, linoleic acid 0.2mg/L, phenol red 12mg/L, putrescine dihydrochloride 5.5mg/L, sodium pyruvate 105mg/L, reduced glutathione 1.7mg/L, galactose 200mg/L, adenine 12.5mg/L, serotonin 5.5mg/L, water-soluble cholesterol 2.8mg/L, guanine hydrochloride 14.5mg/L, thymine 0.2mg/L, uracil 0.2mg/L, thymidine 0.38mg/L, human transferrin 6.5mg/L, recombinant insulin 10mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)2800 mg/L.

As a still further scheme of the invention: the human transferrin is saturated iron human transferrin.

As a still further scheme of the invention: the recombinant insulin is a full-chain recombinant insulin.

Compared with the prior art, the invention has the beneficial effects that:

in the low serum culture medium prepared by the invention and used for ST cell culture and corresponding virus production, the conventional cell culture can be performed on ST cells by replacing the conventional cell culture medium under the condition that 3% special newborn calf serum for swine fever is added to an Ornithogen ST low serum culture medium IV series, and the cell culture state and the number have no obvious difference from those of the conventional culture method, so that the good growth state of the cells is maintained; meanwhile, the culture system can be used for better propagating the classical swine fever virus, and the virus titer is not less than 50 ten thousand times RID.

Drawings

FIG. 1 shows the microscopic observation results of F3 generation cells cultured in MEM.

FIG. 2 shows the result of microscopic observation of LSM cultured cells F3.

FIG. 3 shows that the rabbit reaction in the rabbit assay is a double qualitative thermal reaction in the early stage (61 h).

FIG. 4 shows that the rabbit reaction in the rabbit assay is a double qualitative thermal reaction in the early stage (60 h).

Detailed Description

The technical solution of the present invention will be described in further detail with reference to specific embodiments.