Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine

A PRRS virus, cell suspension technology, applied in animal cells, viruses/phages, vertebrate cells, etc., can solve the problems of limited increase in cell density, high labor intensity, easy pollution, etc., to solve the problem of batch differences , The effect of simplifying the production process and having a high degree of automation

Active Publication Date: 2015-09-09
郑州爱科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Due to the anchorage-dependent growth of the original Marc-145 cells, the production of PRRS vaccines currently mainly adopts the traditional spinner bottle process. Each production batch of vaccine antigen preparation requires hundreds or even thousands of spinner bottles, and each The spinner bottle is a separate culture operation unit. In a production cycle, each spinner bottle needs to go through cell inoculation, virus inoculation, and virus harvesting three times. The content is not the same, which makes the quality of the vaccine unstable and the difference between batches is large; in addition, the use of cell spinner bottle culture technology not only has low production efficiency, but also is easy to pollute and the virus titer is unstable. This traditional technology can no longer meet the large-scale industrialization. Production needs are gradually replaced by automated production
[0004] Due to the anchorage-dependent growth of Marc-145 cells, the current automatic production research of PRRS vaccine is mainly based on carrier-dependent cell suspension culture. The patent publication number is CN102002482B "Production method of porcine respiratory and reproductive disorder syndrome virus" and patent publication number The patent CN102552896B "A Method for Preparing Porcine Reproductive and Respiratory Syndrome Vaccine Using a Bioreactor" all discloses the method for producing PRRS virus by culture of disc carrier cells; Vaccine Method” discloses a method for producing PRRS virus by using the microcarrier cell suspension culture method; although microcarrier and paper carrier culture are highly automated, cell culture is still adherent culture in essence, and the cost of the carrier is relatively high, and most It is a one-time use, and the cell growth is limited by many factors such as the carrier and cannot maximize the growth. The increase in cell density is limited, especially in the aspect of industrial production scale-up. The general culture volume of carrier suspension culture is below 300 liters. This congenital defect limits Large-scale application of the technology

Method used

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  • Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine
  • Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine
  • Monkey embryo renal epithelial cell Marc-145 suspension adapted strain and application thereof in culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of PRRSV vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Expansion and culture of monkey embryonic kidney epithelial cell Marc-145 suspension-adapted strain

[0034] Subculture the Marc-145 suspension-adapted strain of monkey embryonic kidney epithelial cells preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: C201542, and then inoculate it into a bioreactor for suspension culture to obtain a Marc-145 cell suspension culture solution ,Specific steps are as follows:

[0035] ① Take the Marc-145 suspension-adapted strain of monkey embryonic kidney epithelial cells frozen in liquid nitrogen, melt it quickly, add it to a shaker flask filled with nutrient solution, and culture it at 36-37°C for 60-72 hours, according to the ratio of 1:3-1:5 Ratio subculture and amplify culture;

[0036] ② Dilute the Marc-145 cell suspension-adapted strain amplified in step ① to 0.5×10 6 Cells / ml density, inoculated into the bioreactor, the culture temperature is 36-37°C, the pH value is 6.8-...

Embodiment 2

[0040] Embodiment 2 The titer comparison of cultivating PRRS virus with different inoculation doses

[0041] Five 500ml shake flasks were used to culture Marc-145 suspension cells, and the inoculated cells were all 0.58×10 6 cells / ml, cultured for 72 hours under the same culture conditions, the cell density was expanded to more than 3×10 6 Cells / ml, according to the final volume of the culture medium 1.0%, 2.0%, 3.0%, 4.0%, 5.0% inoculated PRRS TJM-92 strain, the viable cell density is lower than 0.5×10 6 Viruses were harvested at 1 / ml. Determination of virus titer (TCID) according to conventional methods 50 / ml), the results are shown in Table 2.

[0042] Table 2

[0043]

Embodiment 3

[0044] Embodiment 3 The comparison of cultivating PRRS virus titer in different culture modes

[0045] Select 2 spinner flasks of well-growing adherent Marc-145 cells, and use 2 500ml shake flasks to culture Marc-145 suspension cells. After the spinner flask cells are covered with a monolayer, the shake flask cell density is greater than 3×10 6 cells / ml, according to 3.0% of the final volume of the culture medium, the PRRS TJM-92 strain was inoculated, and when the CPE of the spinner bottle cells reached 70%, the shaker flask cell density was less than 0.5×10 6 Harvest the virus solution at the time of individual / ml, freeze and thaw twice, measure the virus titer (TCID 50 / ml), the results are shown in Table 3.

[0046] table 3

[0047]

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Abstract

The invention relates to a monkey embryo renal epithelial cell Marc-145 suspension adapted strain and an application thereof in the culture of PRRSV (porcine reproductive and respiratory syndrome virus) and production of a PRRSV vaccine, belonging to the field of biotechnology. The monkey embryo renal epithelial cell Marc-145 suspension adapted strain provided by the invention is collected in the China Center for Type Culture Collection, with a collection number CCTCC No:C201542. The invention also discloses a method for culturing PRRSV by use of the Marc-145 cell suspension adapted strain. By adopting the Marc-145 cell suspension adapted strain provided by the invention, full suspension culture of the Marc-145 cell in a culture medium is realized, and thus the PRRSV is produced by use of the Marc-145 cell suspension adapted strain, step-by-step magnification using a bioreactor is easy to realize in industrial production, and large-scale production of PRRSV vaccine is realized; and compared with the existing spinner-bottle and carrier suspension culture technology, the production technology is simplified, the production cycle is shortened, the production cost is reduced, and the quality and yield of PRRSV vaccine are further improved.

Description

technical field [0001] The invention relates to a monkey embryo kidney epithelial cell Marc-145 suspension adaptation strain and its application in cultivating PRRS virus and producing PRRS virus vaccine, belonging to the field of biotechnology, more specifically, the invention relates to a method using A method for the production of PRRS virus by fully suspending Marc-145 cells in a bioreactor. 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). They grow anchorage-dependently and can be continuously subcultured without tumorigenicity. 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 PRRS vaccines. [0003] Due to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
Inventor 李少英徐树兰
Owner 郑州爱科生物科技有限公司
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