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Serum-free culture media and application thereof as well as culture method of classical swine fever virus

A serum-free culture medium and swine fever virus technology, applied in the field of serum-free culture and swine fever virus culture, can solve the problems of inability to directly learn from and great differences in culture conditions, and achieve good amino acid metabolism, fast growth rate, Rapid proliferation effect

Active Publication Date: 2015-01-14
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since the culture conditions required by different cells or viruses are very different, the inventors cannot directly learn from the composition of the existing serum-free medium, and must address this problem, re-study, and obtain suitable for ST cell culture, and CSFV Serum-free medium for proliferation

Method used

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  • Serum-free culture media and application thereof as well as culture method of classical swine fever virus
  • Serum-free culture media and application thereof as well as culture method of classical swine fever virus
  • Serum-free culture media and application thereof as well as culture method of classical swine fever virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0146] Dissolve the following substances respectively, mix each solution, and stir in the dark for 30 minutes until fully mixed, then add 800ml of ultra-clean pure water without pyrogen, stir and mix again, set the volume to 1000ml, and adjust the pH of the solution to 7.2. Added substances include: MEM basal medium 9.8g; sodium pyruvate 50-200mg; soybean protein hydrolyzate 0.1-2g; cholesterol 0.1-6mg; basic fibroblast growth factor 0.5-50mg; ;Insulin 5~50mg; L-alanine 2~30mg; L-valine 52~150mg; L-leucine 10~80mg; L-isoleucine 5~180mg; L-methionine 15 ~300mg; L-proline

[0147] 10~250mg; L-Phenylalanine 10~250mg; L-Tryptophan 5~300mg; L-Glycine

[0148] 0~50mg; L-serine 5~150mg; L-threonine 5~250mg; L-cysteine ​​5~250mg; L-asparagine 5~200mg; L-glutamine 250~400mg; L- Tyrosine 5~250mg; L-Aspartic Acid 2~80mg; L-Glutamic Acid 5~200mg; L-Lysine 6~100mg; L-Arginine

[0149] 5~300mg; L-histidine 5~300mg; Hydroxyproline 0~40mg; Myristic acid 0.01~0.3mg; Palmitic acid 0.01~0.3mg...

Embodiment 2

[0153] According to the mode in Example 1, prepare the first kind of target serum-free medium, wherein each substance is as shown in Table 1,

[0154] Table 1 Components of serum-free medium suitable for ST cell growth

[0155] MEM basal medium 9.8g / L sodium pyruvate 200mg / L Soy Protein Hydrolyzate 2g / L cholesterol 2.5mg / L basic fibroblast growth factor 25mg / L Inhibin B 25mg / L insulin 25mg / L L-alanine 20mg / L L-valine 150mg / L L-leucine 45mg / L L-isoleucine 58mg / L L-methionine 150mg / L L-proline 150mg / L L-phenylalanine 150mg / L L-tryptophan 50mg / L

[0156] L-Glycine 10mg / L L-serine 55mg / L L-threonine 55mg / L L-cysteine 55mg / L L-Asparagine 50mg / L L-Glutamine 250mg / L L-tyrosine 55mg / L L-Aspartic Acid 20mg / L L-glutamic acid 50mg / L L-Lysine 60mg / L L-Arginine 50mg / L L-histidine 50mg / L ...

Embodiment 3

[0158] Dissolve the following substances respectively, mix each solution, and stir in the dark for 30 minutes until fully mixed, then add 800ml of ultra-clean pure water without pyrogen, stir and mix again, set the volume to 1000ml, and adjust the pH of the solution to 6.6. Added substances include: MEM basal medium 9.8g; sodium pyruvate 50-300mg; soybean protein hydrolyzate 0.1-2g; cholesterol 2-6mg; basic fibroblast growth factor 0.5-20mg; ;Insulin 10~50mg; L-alanine 10~100mg; L-valine

[0159] 2~35mg; L-methionine 10~30mg; L-proline 10~250mg; L-tryptophan 5~300mg; L-glycine 10~50mg; L-serine 5~150mg; L-cysteine Amino acid 5~250mg; L-Asparagine 5~200mg; L-Glutamine 100~200mg; L-Aspartic Acid 2~80mg; L-Glutamic Acid 5~200mg;

[0160] Hydroxyproline 10~60mg; Myristic acid 0.01~0.3mg; Palmitic acid 0.01~0.3mg; Palmitoleic acid 0.01~0.3mg; Stearic acid 0.01~0.3mg; Spermine 0.1~0.3ml; Spermidine 0.1~ 0.3ml; reduced glutathione 3~15mg; ethanolamine 1.5~3ml; β-mercaptoethanol 1~2...

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Abstract

The invention relates to the biotechnical field, particularly relates to the technical field of animal biological product engineering and more particularly relates to serum-free culture media and application thereof as well as a culture method of a classical swine fever virus by using the culture media. According to the preparation method of the serum-free culture media, with an MEM (minimum essential medium) as a base culture medium, based on research on growth and multiplication characteristics of a swine testis (ST) cell and the classical swine fever virus, by adding different components into the base culture medium, a series of swine testis cell serum-free culture media as well as classical swine fever virus multiplication serum-free culture media for different growth stages are obtained respectively; therefore, possible pollution risks of BVDV (bovine viral diarrhea virus) and an antibody thereof are avoided, cell adherence growth of the ST cell can be realized at the same time; and moreover, the ST cell can grow quickly, has an intact cell side, is full in individual and is capable of being sub-cultured for a long time.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the technical field of veterinary biological products engineering, more specifically to the serum-free culture and its application and the culture method of swine fever virus using these medium. Background technique [0002] Classical swine fever (CSF) is a highly contagious disease caused by classical swine fever virus (CSFV), and is listed as a category A zoonotic disease by the World Organization for Animal Health. At present, the main means of controlling swine fever epidemic disease in my country is to inoculate the live vaccine of classical swine fever lanovirus (HCLV). The traditional production method of swine fever vaccine is to infect rabbits, and then collect spleen and peritoneal lymph to make tissue vaccines, or use primary bovine testicular cells to proliferate cell vaccines. However, the above two methods have been gradually replaced by the continuous passage ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12R1/93
Inventor 冯磊褚轩吴培培王伟峰侯继波华涛陈丽
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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