CN110643568A

CN110643568A - Low-serum culture medium for BHK-21 cell culture and corresponding virus production

Info

Publication number: CN110643568A
Application number: CN201910900397.2A
Authority: CN
Inventors: 房圆瑗; 齐智; 刘海英; 巩俊廷
Original assignee: Shandong Oracle Biotechnology Co Ltd
Current assignee: Shandong Oracle Biotechnology Co Ltd
Priority date: 2019-09-23
Filing date: 2019-09-23
Publication date: 2020-01-03

Abstract

The invention discloses a low serum culture medium for BHK-21 cell culture and corresponding virus production, which comprises the following components: amino acids, vitamins, inorganic salts, proteins, trace elements and auxiliary components; the amino acid comprises glycine, L-arginine hydrochloride, L-asparagine, L-glutamic acid, L-glutamine, L-lysine hydrochloride and L-serine; the protein comprises 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), sodium dihydrogen phosphate monohydrate, milk protein hydrolysate, bovine serum albumin and recombinant human insulin; the low serum culture medium for BHK-21 cell culture and corresponding virus production does not need to be added with serum in the maintenance stage of producing the pseudorabies vaccine by using the BHK-21 cells, the serum consumption is further reduced, the good growth state of the cells can be still kept, the virus titer is improved, the reduction amplitude of the virus price of a semi-finished product is small, and the storage time of the semi-finished product is prolonged.

Description

Low-serum culture medium for BHK-21 cell culture and corresponding virus production

Technical Field

The invention relates to the field of production of biopharmaceutical vaccines, in particular to a low serum culture medium for BHK-21 cell culture and production of corresponding viruses.

Background

BHK cells refer to juvenile Syrian rat Kidney cells (BabyHamster Syrian Kidney cell) established in 1961. The original cell line was fibroblast, anchorage dependent. Single cell clonal cells were obtained in 1963. After numerous passages, the cell can grow in suspension, and it is widely used for proliferating various viruses and producing veterinary vaccines.

The traditional BHK-21 cell culture mode is that DMEM high-sugar medium is added with 8% calf serum to culture cells, the serum consumption is large, and the cost is high; the titer of the pseudorabies vaccine semi-finished product produced by the traditional method is between 7.0 and 8.0, the toxin price is reduced quickly under the freezing condition, the storage time is short, and the semi-finished product meeting the mating standard is difficult to obtain.

Some low-serum cell culture products are on the market, and although the problem of serum dosage is solved, the problem of rapid reduction of the virus price of a pseudorabies vaccine semi-finished product is still not solved, and based on the problem, a low-serum culture medium for BHK-21 cell culture and corresponding virus production is provided.

Disclosure of Invention

The invention aims to provide a low-serum culture medium for BHK-21 cell culture and corresponding virus production, so as to solve the problems in the background technology.

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

a low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: amino acids, vitamins, inorganic salts, proteins, trace elements and auxiliary components; the amino acids comprise 10-50mg/L, L-alanine 5-15mg/L, L-arginine hydrochloride 50-150mg/L, L-asparagine 250-350mg/L, L-aspartic acid 10-15mg/L, L-cystine dihydrochloride 50-120mg/L, cysteine 45-110mg/L, L-glutamic acid 100-200mg/L, L-glutamine 200-400mg/L, L-histidine hydrochloride monohydrate 20-50mg/L, L-isoleucine 50-120mg/L, L-leucine 50-120mg/L, L-lysine hydrochloride 100-200mg/L, L-methionine 20-50mg/L, and, L-phenylalanine 50-100mg/L, L-proline 5-15mg/L, L-serine 100-200mg/L, L-threonine 50-120mg/L, L-tryptophan 10-20mg/L, L-tyrosine disodium dihydrate 50-120mg/L, L-valine 50-120 mg/L; the protein comprises 1500mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) 1000-.

As a further scheme of the invention: the auxiliary components comprise 5000mg/L of D-glucose 2000-plus, 0.5-3mg/L of ethanolamine, 0.5-3mg/L of reduced glutathione, 5-15mg/L of phenol red, 50-150mg/L of sodium pyruvate, 0.1-0.5mg/L of thymidine, 0.1-0.5mg/L of adenine, 1-5mg/L of sodium hypoxanthine, 0.02-0.1mg/L of linoleic acid, 0.05-0.15mg/L of lipoic acid and 0.05-0.15mg/L of putrescine dihydrochloride.

As a still further scheme of the invention: the microelements comprise ammonium metavanadate 0.0001-0.0005mg/L, copper sulfate pentahydrate 0.0005-0.0015mg/L, manganese dichloride 0.00001-0.00006mg/L, and sodium selenite 0.002-0.006 mg/L.

As a still further scheme of the invention: the vitamins include ascorbic acid phosphate 1-3mg/L, choline chloride 2-5mg/L, D-calcium pantothenate 2-5mg/L, folic acid 2-5mg/L, nicotinamide 2-5mg/L, pyridoxine hydrochloride 2-5mg/L, riboflavin 0.1-1mg/L, thiamine hydrochloride 2-5mg/L, inositol 5-10mg/L, biotin 0.01-0.04mg/L, B12 vitamin 2-5 mg/L.

As a still further scheme of the invention: the inorganic salts comprise 100-300mg/L of anhydrous calcium chloride, 0.05-0.2mg/L of ferric nitrate nonahydrate, 50-100mg/L of anhydrous magnesium sulfate, 500mg/L of potassium chloride 300 and 6000mg/L of sodium chloride 4000.

As a still further scheme of the invention: comprises the following components: glycine 37.5mg/L, L-alanine 8.9mg/L, L-arginine hydrochloride 84mg/L, L-asparagine 256.8mg/L, L-aspartic acid 13.3mg/L, L-cystine dihydrochloride 63mg/L, cysteine 99mg/L, L-glutamic acid 195.3mg/L, L-glutamine 398.8mg/L, L-histidine hydrochloride monohydrate 42mg/L, L-isoleucine 105mg/L, L-leucine 105mg/L, L-lysine hydrochloride 146mg/L, L-methionine 30mg/L, L-phenylalanine 66mg/L, L-proline 11.5mg/L, L-serine.167 mg/L, L-threonine 95mg/L, 16mg/L, L-tyrosine disodium dihydrate 104mg/L, L-valine 94mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1287mg/L, monobasic sodium phosphate monohydrate 125mg/L, milk protein hydrolysate 400mg/L, bovine serum albumin 200mg/L, bovine transferrin 7.5mg/L, recombinant human insulin 12.5mg/L, ammonium metavanadate 3.00E-04mg/L, copper sulfate pentahydrate 0.00125mg/L, manganese dichloride 5.00E-05mg/L, sodium selenite 0.005mg/L, D-glucose 4500mg/L, ethanolamine 1.9mg/L, reduced glutathione 2mg/L, phenol red 8.1mg/L, sodium pyruvate 110mg/L, thymidine 0.365mg/L, Adenine 0.365mg/L, hypoxanthine sodium 2.39mg/L, linoleic acid 0.042mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.081mg/L, ascorbic acid phosphate 2.5mg/L, choline chloride 4mg/L, D-calcium pantothenate 4mg/L, folic acid 4mg/L, nicotinamide 4mg/L, pyridoxine hydrochloride 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, inositol 7.2mg/L, biotin 0.035mg/L, B12 vitamin 2.72mg/L, anhydrous calcium chloride 200mg/L, ferric nitrate nonahydrate 0.1mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400mg/L, sodium chloride 5000 mg/L.

As a still further scheme of the invention: the D-glucose is anhydrous glucose

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

the low serum culture medium for BHK-21 cell culture and corresponding virus production does not need to be added with serum in the maintenance stage of producing the pseudorabies vaccine by using the BHK-21 cells, the serum consumption is further reduced, the good growth state of the cells can be still kept, the virus titer is improved, the reduction amplitude of the virus price of a semi-finished product is small, and the storage time of the semi-finished product is prolonged; meanwhile, the low serum culture medium for BHK-21 cell culture and corresponding virus production has the characteristics of fast cell growth, stable pH value, suitability for low-density culture and the like, can reduce the cost, improve the efficiency, ensure stable and sustainable production quality in practical production application, and can meet the requirement of large-scale PRV production.

Detailed Description

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

Example 1

A low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: glycine 37.5mg/L, L-alanine 8.9mg/L, L-arginine hydrochloride 84mg/L, L-asparagine 256.8mg/L, L-aspartic acid 13.3mg/L, L-cystine dihydrochloride 63mg/L, cysteine 99mg/L, L-glutamic acid 195.3mg/L, L-glutamine 398.8mg/L, L-histidine hydrochloride monohydrate 42mg/L, L-isoleucine 105mg/L, L-leucine 105mg/L, L-lysine hydrochloride 146mg/L, L-methionine 30mg/L, L-phenylalanine 66mg/L, L-proline 11.5mg/L, L-serine.167 mg/L, L-threonine 95mg/L, 16mg/L, L-tyrosine disodium dihydrate 104mg/L, L-valine 94mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1287mg/L, monobasic sodium phosphate monohydrate 125mg/L, milk protein hydrolysate 400mg/L, bovine serum albumin 200mg/L, bovine transferrin 7.5mg/L, recombinant human insulin 12.5mg/L, ammonium metavanadate 3.00E-04mg/L, copper sulfate pentahydrate 0.00125mg/L, manganese dichloride 5.00E-05mg/L, sodium selenite 0.005mg/L, D-glucose 4500mg/L, ethanolamine 1.9mg/L, reduced glutathione 2mg/L, phenol red 8.1mg/L, sodium pyruvate 110mg/L, thymidine 0.365mg/L, Adenine 0.365mg/L, hypoxanthine sodium 2.39mg/L, linoleic acid 0.042mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.081mg/L, ascorbic acid phosphate 2.5mg/L, choline chloride 4mg/L, D-calcium pantothenate 4mg/L, folic acid 4mg/L, nicotinamide 4mg/L, pyridoxine hydrochloride 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, inositol 7.2mg/L, biotin 0.035mg/L, B12 vitamin 2.72mg/L, anhydrous calcium chloride 200mg/L, ferric nitrate nonahydrate 0.1mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400mg/L, sodium chloride 5000 mg/L.

Wherein the D-glucose is anhydrous glucose.

Example 2

A low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: glycine 10mg/L, L-alanine 5mg/L, L-arginine hydrochloride 50mg/L, L-asparagine 250mg/L, L-aspartic acid 10mg/L, L-cystine dihydrochloride 50mg/L, cysteine 45mg/L, L-glutamic acid 100mg/L, L-glutamine 200mg/L, L-histidine hydrochloride monohydrate 20mg/L, L-isoleucine 50mg/L, L-leucine 50mg/L, L-lysine hydrochloride 100mg/L, L-methionine 20mg/L, L-phenylalanine 50mg/L, L-proline 5mg/L, L-serine 100mg/L, L-threonine 50mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 50mg/L, L-valine 50mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1000mg/L, sodium dihydrogen phosphate monohydrate 50mg/L, milk protein hydrolysate 50mg/L, bovine serum albumin 100mg/L, bovine transferrin 5mg/L, recombinant human insulin 5mg/L, ammonium metavanadate 0.0001mg/L, copper sulfate pentahydrate 0.0005mg/L, manganese dichloride 0.00001mg/L, sodium selenite 0.002mg/L, D-glucose 2000mg/L, ethanolamine 0.5mg/L, reduced glutathione 0.5mg/L, phenol red 5mg/L, sodium pyruvate 50mg/L, thymidine 0.1mg/L, adenine 0.1mg/L, Hypoxanthine sodium 1mg/L, linoleic acid 0.02mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.05mg/L, ascorbic acid phosphate 1mg/L, choline chloride 2mg/L, D-calcium pantothenate 2mg/L, folic acid 2mg/L, nicotinamide 2mg/L, pyridoxine hydrochloride 2mg/L, riboflavin 0.1mg/L, thiamine hydrochloride 2mg/L, inositol 5mg/L, biotin 0.01mg/L, B12 vitamin 2mg/L, anhydrous calcium chloride 100mg/L, ferric nitrate nonahydrate 0.05mg/L, anhydrous magnesium sulfate 50mg/L, potassium chloride 300mg/L, and sodium chloride 4000 mg/L.

Wherein the D-glucose is anhydrous glucose.

Example 3

A low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: glycine 50mg/L, L-alanine 15mg/L, L-arginine hydrochloride 150mg/L, L-asparagine 350mg/L, L-aspartic acid 15mg/L, L-cystine 120mg/L, cysteine 110mg/L, L-glutamic acid 200mg/L, L-glutamine 400mg/L, L-histidine hydrochloride monohydrate 50mg/L, L-isoleucine 120mg/L, L-leucine 120mg/L, L-lysine hydrochloride 200mg/L, L-methionine 50mg/L, L-phenylalanine 100mg/L, L-proline 15mg/L, L-serine 200mg/L, L-threonine 120mg/L, L-tryptophan 20mg/L, L-tyrosine disodium dihydrate 120mg/L, L-valine 120mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1500mg/L, monobasic sodium phosphate monohydrate 150mg/L, milk protein hydrolysate 600mg/L, bovine serum albumin 600mg/L, bovine transferrin 15mg/L, recombinant human insulin 15mg/L, ammonium metavanadate 0.0005mg/L, copper sulfate pentahydrate 0.0015mg/L, manganese dichloride 0.00006mg/L, sodium selenite 0.006mg/L, D-glucose 5000mg/L, ethanolamine 3mg/L, reduced glutathione 3mg/L, phenol red 15mg/L, sodium pyruvate 150mg/L, thymidine 0.5mg/L, adenine 0.5mg/L, 5mg/L of hypoxanthine sodium, 0.1mg/L of linoleic acid, 0.15mg/L of lipoic acid, 0.15mg/L of putrescine dihydrochloride, 3mg/L of ascorbic acid phosphate, 5mg/L of choline chloride, 5mg/L, D-calcium pantothenate, 5mg/L of folic acid, 5mg/L of nicotinamide, 5mg/L of pyridoxine hydrochloride, 1mg/L of riboflavin, 5mg/L of thiamine hydrochloride, 10mg/L of inositol, 0.04mg/L, B12 of vitamin, 300mg/L of anhydrous calcium chloride, 0.2mg/L of ferric nitrate nonahydrate, 100mg/L of anhydrous magnesium sulfate, 500mg/L of potassium chloride and 6000mg/L of sodium chloride.

Wherein the D-glucose is anhydrous glucose.

Example 4

A low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: glycine 10mg/L, L-alanine 5mg/L, L-arginine hydrochloride 50mg/L, L-asparagine 250mg/L, L-aspartic acid 10mg/L, L-cystine dihydrochloride 50mg/L, cysteine 45mg/L, L-glutamic acid 100mg/L, L-glutamine 200mg/L, L-histidine hydrochloride monohydrate 20mg/L, L-isoleucine 50mg/L, L-leucine 50mg/L, L-lysine hydrochloride 100mg/L, L-methionine 20mg/L, L-phenylalanine 50mg/L, L-proline 5mg/L, L-serine 100mg/L, L-threonine 50mg/L, L-tryptophan 10mg/L, L-tyrosine disodium dihydrate 50mg/L, L-valine 50mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1000mg/L, sodium dihydrogen phosphate monohydrate 50mg/L, milk protein hydrolysate 50mg/L, bovine serum albumin 100mg/L, bovine transferrin 5mg/L, recombinant human insulin 5mg/L, ammonium metavanadate 3.00E-04mg/L, copper sulfate pentahydrate 0.00125mg/L, manganese dichloride 5.00E-05mg/L, sodium selenite 0.005mg/L, D-glucose 4500mg/L, ethanolamine 1.9mg/L, reduced glutathione 2mg/L, phenol red 8.1mg/L, sodium pyruvate 110mg/L, thymidine 0.365mg/L, Adenine 0.365mg/L, hypoxanthine sodium 2.39mg/L, linoleic acid 0.042mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.081mg/L, ascorbic acid phosphate 2.5mg/L, choline chloride 4mg/L, D-calcium pantothenate 4mg/L, folic acid 4mg/L, nicotinamide 4mg/L, pyridoxine hydrochloride 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, inositol 7.2mg/L, biotin 0.035mg/L, B12 vitamin 2.72mg/L, anhydrous calcium chloride 200mg/L, ferric nitrate nonahydrate 0.1mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400mg/L, sodium chloride 5000 mg/L.

Wherein the D-glucose is anhydrous glucose.

Example 5

A low serum culture medium for BHK-21 cell culture and production of corresponding viruses comprises the following components: glycine 50mg/L, L-alanine 15mg/L, L-arginine hydrochloride 150mg/L, L-asparagine 350mg/L, L-aspartic acid 15mg/L, L-cystine 120mg/L, cysteine 110mg/L, L-glutamic acid 200mg/L, L-glutamine 400mg/L, L-histidine hydrochloride monohydrate 50mg/L, L-isoleucine 120mg/L, L-leucine 120mg/L, L-lysine hydrochloride 200mg/L, L-methionine 50mg/L, L-phenylalanine 100mg/L, L-proline 15mg/L, L-serine 200mg/L, L-threonine 120mg/L, L-tryptophan 20mg/L, L-tyrosine disodium dihydrate 120mg/L, L-valine 120mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES)1000mg/L, sodium dihydrogen phosphate monohydrate 50mg/L, milk protein hydrolysate 50mg/L, bovine serum albumin 100mg/L, bovine transferrin 5mg/L, recombinant human insulin 5mg/L, ammonium metavanadate 0.0001mg/L, copper sulfate pentahydrate 0.0005mg/L, manganese dichloride 0.00001mg/L, sodium selenite 0.002mg/L, D-glucose 2000mg/L, ethanolamine 0.5mg/L, reduced glutathione 0.5mg/L, phenol red 5mg/L, sodium pyruvate 50mg/L, thymidine 0.1mg/L, adenine 0.1mg/L, Hypoxanthine sodium 1mg/L, linoleic acid 0.02mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.05mg/L, ascorbic acid phosphate 1mg/L, choline chloride 2mg/L, D-calcium pantothenate 2mg/L, folic acid 2mg/L, nicotinamide 2mg/L, pyridoxine hydrochloride 2mg/L, riboflavin 0.1mg/L, thiamine hydrochloride 2mg/L, inositol 5mg/L, biotin 0.01mg/L, B12 vitamin 2mg/L, anhydrous calcium chloride 100mg/L, ferric nitrate nonahydrate 0.05mg/L, anhydrous magnesium sulfate 50mg/L, potassium chloride 300mg/L, and sodium chloride 4000 mg/L.

Wherein the D-glucose is anhydrous glucose.

PRV Pilot-plant test for BHK-21 cell proliferation

The experimental procedure was as follows:

1) culturing a cell monolayer of the subcultured domesticated low serum adaptive cell strain BHK-LSA by using growth medium, and inoculating 2.5 multiplied by 108cells in each rotary bottle;

2) adding BHK-SLM + 2% serum growth liquid about 300m into each rotary bottle;

3) carrying out rotary culture at 37 ℃ in a greenhouse at 12 rpm for 72 h;

4) taking well-propagated BHK-LSA cells, discarding growth medium, inoculating pseudorabies virus according to a proper volume ratio, and adsorbing at 37 ℃ for 60 minutes. And (3) supplementing 300ml of maintenance solution after adsorption is finished, placing in a greenhouse for culturing for 24-36h, and harvesting the virus when the cytopathic effect reaches more than 80%. When the quality of the virus seeds is excellent, the virus seeds can be collected within 24 h;

5) freezing and thawing for 1 time after the virus is harvested; and mixing the viruses in the bottles, and sampling for detection.

Results of the experiment

According to the experimental results that the low serum culture medium of the periomethyl gold BHK-21 used in the pilot test is used for proliferating the PRV after the transfer bottle passage, no serum is added in the maintenance stage, the harvesting time is short, and the three batches of toxic prices exceed the estimated range, the experimental results show that: the low serum culture medium for BHK-21 cell culture and corresponding virus production has the characteristics of fast cell growth, stable pH value, suitability for low-density culture and the like, and simultaneously shows that the low serum culture medium can reduce the cost, improve the efficiency, ensure the stable and sustainable production quality and meet the requirement of large-scale PRV production in practical production application.

The low serum culture medium for BHK-21 cell culture and corresponding virus production does not need to be added with serum in the maintenance stage of producing the pseudorabies vaccine by using the BHK-21 cells, the serum consumption is further reduced, the good growth state of the cells can be still kept, the virus titer is improved, the reduction amplitude of the virus price of a semi-finished product is small, and the storage time of the semi-finished product is prolonged.

While the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art.

Claims

1. A low serum culture medium for BHK-21 cell culture and production of corresponding viruses is characterized by comprising the following components: amino acids, vitamins, inorganic salts, proteins, trace elements and auxiliary components; the amino acids comprise 10-50mg/L, L-alanine 5-15mg/L, L-arginine hydrochloride 50-150mg/L, L-asparagine 250-350mg/L, L-aspartic acid 10-15mg/L, L-cystine dihydrochloride 50-120mg/L, cysteine 45-110mg/L, L-glutamic acid 100-200mg/L, L-glutamine 200-400mg/L, L-histidine hydrochloride monohydrate 20-50mg/L, L-isoleucine 50-120mg/L, L-leucine 50-120mg/L, L-lysine hydrochloride 100-200mg/L, L-methionine 20-50mg/L, and, L-phenylalanine 50-100mg/L, L-proline 5-15mg/L, L-serine 100-200mg/L, L-threonine 50-120mg/L, L-tryptophan 10-20mg/L, L-tyrosine disodium dihydrate 50-120mg/L, L-valine 50-120 mg/L; the protein comprises 1500mg/L of 4-hydroxyethyl piperazine ethanesulfonic acid 1000-.

2. The low serum culture medium for BHK-21 cell culture and production of corresponding viruses of claim 1, wherein the auxiliary components comprise 5000mg/L of D-glucose 2000-.

3. The low serum culture medium for BHK-21 cell culture and production of corresponding viruses of claim 2, wherein the trace elements comprise ammonium metavanadate 0.0001-0.0005mg/L, copper sulfate pentahydrate 0.0005-0.0015mg/L, manganese dichloride 0.00001-0.00006mg/L, sodium selenite 0.002-0.006 mg/L.

4. The low serum culture medium for BHK-21 cell culture and corresponding virus production of claim 3, wherein the vitamins include ascorbic acid phosphate 1-3mg/L, choline chloride 2-5mg/L, D-calcium pantothenate 2-5mg/L, folic acid 2-5mg/L, nicotinamide 2-5mg/L, pyridoxine hydrochloride 2-5mg/L, riboflavin 0.1-1mg/L, thiamine hydrochloride 2-5mg/L, inositol 5-10mg/L, biotin 0.01-0.04mg/L, B12 vitamin 2-5 mg/L.

5. The low serum culture medium for BHK-21 cell culture and corresponding virus production according to claim 4, wherein the inorganic salts comprise anhydrous calcium chloride 100-300mg/L, ferric nitrate nonahydrate 0.05-0.2mg/L, anhydrous magnesium sulfate 50-100mg/L, potassium chloride 300-500mg/L, sodium chloride 4000-6000 mg/L.

6. The low serum medium for BHK-21 cell culture and production of corresponding viruses according to claim 5, comprising the following components: glycine 37.5mg/L, L-alanine 8.9mg/L, L-arginine hydrochloride 84mg/L, L-asparagine 256.8mg/L, L-aspartic acid 13.3mg/L, L-cystine dihydrochloride 63mg/L, cysteine 99mg/L, L-glutamic acid 195.3mg/L, L-glutamine 398.8mg/L, L-histidine hydrochloride monohydrate 42mg/L, L-isoleucine 105mg/L, L-leucine 105mg/L, L-lysine hydrochloride 146mg/L, L-methionine 30mg/L, L-phenylalanine 66mg/L, L-proline 11.5mg/L, L-serine.167 mg/L, L-threonine 95mg/L, 16mg/L, L-tyrosine disodium dihydrate 104mg/L, L-valine 94mg/L, 4-hydroxyethyl piperazine ethanesulfonic acid 1287mg/L, sodium dihydrogen phosphate monohydrate 125mg/L, milk protein hydrolysate 400mg/L, bovine serum albumin 200mg/L, bovine transferrin 7.5mg/L, recombinant human insulin 12.5mg/L, ammonium metavanadate 3.00E-04mg/L, copper sulfate pentahydrate 0.00125mg/L, manganese dichloride 5.00E-05mg/L, sodium selenite 0.005mg/L, D-glucose 4500mg/L, ethanolamine 1.9mg/L, glutathione reduced form 2mg/L, phenol red 8.1mg/L, sodium pyruvate 110mg/L, thymidine 0.365mg/L, Adenine 0.365mg/L, hypoxanthine sodium 2.39mg/L, linoleic acid 0.042mg/L, lipoic acid 0.105mg/L, putrescine dihydrochloride 0.081mg/L, ascorbic acid phosphate 2.5mg/L, choline chloride 4mg/L, D-calcium pantothenate 4mg/L, folic acid 4mg/L, nicotinamide 4mg/L, pyridoxine hydrochloride 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, inositol 7.2mg/L, biotin 0.035mg/L, B12 vitamin 2.72mg/L, anhydrous calcium chloride 200mg/L, ferric nitrate nonahydrate 0.1mg/L, anhydrous magnesium sulfate 97.67mg/L, potassium chloride 400mg/L, sodium chloride 5000 mg/L.

7. The low serum medium for BHK-21 cell culture and production of corresponding viruses of claim 2, wherein the D-glucose is anhydrous glucose.