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Method for directly culturing porcine reproductive and respiratory syndrome virus vaccine by using Marc-145 full-suspension cells

A porcine blue-ear disease virus, full-suspension technology, applied in vaccines, viruses, embryonic cells, etc., can solve the problems of inability to produce high-titer antibodies, infection of porcine blue-ear virus, and long neutralizing antibody time

Pending Publication Date: 2019-10-25
BEIJING VBIOSCI INC +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the main method of prevention and treatment of PRRS is vaccination, but in practice, vaccines often fail to prevent PRRS. The main reason is that it takes a long time for the body to produce neutralizing antibodies when the vaccine is immunized. Or can not produce high-titer antibodies, and during this period may be infected with porcine blue ear virus

Method used

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  • Method for directly culturing porcine reproductive and respiratory syndrome virus vaccine by using Marc-145 full-suspension cells
  • Method for directly culturing porcine reproductive and respiratory syndrome virus vaccine by using Marc-145 full-suspension cells
  • Method for directly culturing porcine reproductive and respiratory syndrome virus vaccine by using Marc-145 full-suspension cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Use MEM medium containing 7% fetal bovine serum to resuscitate Marc-145 cryopreserved adherent cells, subculture after the cells grow dense, and subculture twice continuously;

[0035] Use trypsin to digest Marc-145 cells and centrifuge at low speed. The obtained cells are cultured in a serum-free suspension medium with 10% fetal bovine serum at a speed of 40r / min, and the inoculation density is 1.0×10 6 cells / ml for continuous subculture for 4 times;

[0036] Serum-free suspension medium was added with 4% fetal bovine serum to carry out shaker culture at a speed of 60r / min, and the inoculation density was 0.8×10 6 cells / ml for continuous subculture for 4 times;

[0037] Serum-free suspension medium and 1% fetal bovine serum were added to carry out shaker culture at a speed of 80r / min, and the inoculation density was 0.6×10 6 cells / ml for continuous subculture for 4 times;

[0038]The serum-free suspension medium was cultured on a shaker at a rotational speed of 100r...

Embodiment 2

[0046] Use MEM medium containing 9% fetal bovine serum to resuscitate Marc-145 cryopreserved adherent cells, subculture after the cells grow densely, and subculture twice continuously;

[0047] Use trypsin to digest Marc-145 cells and centrifuge at low speed. The obtained cells are cultured in a serum-free suspension medium with 9% fetal bovine serum at a speed of 50r / min, and the seeding density is 1.2×10 6 cells / ml for continuous subculture for 4 times;

[0048] Serum-free suspension medium was added with 5% fetal bovine serum for shaker culture at a speed of 70r / min, and the inoculation density was 1.0×10 6 cells / ml for continuous subculture for 4 times;

[0049] Serum-free suspension medium and 1.5% fetal bovine serum were added to carry out shaker culture at a speed of 90r / min, and the inoculation density was 0.7×10 6 cells / ml for continuous subculture for 4 times;

[0050] The serum-free suspension medium was cultured on a shaker at a speed of 130r / min, and the inocul...

Embodiment 3

[0058] Use MEM medium containing 10% fetal bovine serum to resuscitate Marc-145 cryopreserved adherent cells, subculture after the cells grow densely, and subculture twice continuously;

[0059] Use trypsin to digest Marc-145 cells and centrifuge at low speed. The obtained cells are cultured on a shaker with a serum-free suspension medium and 10% fetal bovine serum at a speed of 60 r / min. The seeding density is 1.5×10 6 cells / ml for continuous subculture for 4 times;

[0060] Serum-free suspension medium and 6% fetal bovine serum were added to carry out shaker culture at a speed of 80r / min, and the inoculation density was 1.2×10 6 cells / ml for continuous subculture for 4 times;

[0061] Serum-free suspension medium was added with 2% fetal bovine serum for shaker culture at a rotational speed of 100r / min, and the inoculation density was 0.8×10 6 cells / ml for continuous subculture for 4 times;

[0062] The serum-free suspension medium was cultured on a shaker at a speed of 14...

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Abstract

The invention relates to the field of porcine reproductive and respiratory syndrome virus vaccine preparation, in particular to a method for directly culturing a porcine reproductive and respiratory syndrome virus vaccine by using Marc-145 full-suspension cells. The invention discloses a method for domesticating Marc-145 monolayer cells into suspension culture. Full-suspension culture of the Marc-145 cells in a culture medium is achieved, then a porcine reproductive and respiratory syndrome virus is directly inoculated into the Marc-145 suspension cells for culture, culture is enlarged by using a bioreactor, the cultured porcine reproductive and respiratory syndrome virus vaccine is stable in proliferation speed, and the virus content is larger than or equal to 108.5 TCID50; a virus liquidis obtained when the vaccine is cultured until the cytopathy reaches 80% or above, and the vaccine is obtained. According to the method, the porcine reproductive and respiratory syndrome virus is directly inoculated into the Marc-145 suspension cells for domestication and culture, and the stability, continuity and safety of the produced suspension virus are effectively improved; besides, the bioreactor is used for culture enlarging, the virus content of each 0.1 ml is larger than or equal to 108.5 TCID50, and the quality and yield of the porcine reproductive and respiratory syndrome vaccine are further improved.

Description

technical field [0001] The invention relates to the technical field of veterinary biopharmaceuticals, in particular to a method for directly cultivating porcine PRRS virus vaccine with Marc-145 full suspension cells. Background technique [0002] Marc-145 cells are monkey embryonic kidney epithelial cells, which are derived from monkey kidney cells and are clones of mother cells (MA104 cells). Suspension-adapted strains can be obtained through suspension adaptation, which can grow in suspension without relying on the carrier. The cells are particularly sensitive to porcine reproductive and respiratory syndrome virus (PRRSV) and are currently widely used in the production of porcine PRRS vaccines. [0003] Porcine blue ear disease (PRRS), also known as mysterious swine disease, new swine disease, porcine epidemic abortion and respiratory syndrome, porcine reproductive and respiratory syndrome, porcine blue ear disease, swine fever, etc., is the most serious swine disease in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/02C12N7/00C12N7/02C12N7/06A61K39/12A61P31/14
CPCA61K39/12A61K2039/5252A61K2039/552A61P31/14C12N5/0603C12N7/00C12N2500/90C12N2770/10034C12N2770/10051C12N2770/10063
Inventor 侯野邵瑞华马玉鑫周佳彬韩丹宋瑞卿
Owner BEIJING VBIOSCI INC
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