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Construction and application of PK-15 stable cell line

A cell line and cell technology, applied in the construction of PK-15 stable cell line, can solve the problem of low virus content and achieve the effect of strong genetic stability

Pending Publication Date: 2019-11-15
成都史纪生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the propagation of PRRSV in Marc-145 cells has the limitation that the virus content is not high. Therefore, increasing the virus content of PRRSV in vitro is one of the important ways to improve the quality of related vaccine products.

Method used

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  • Construction and application of PK-15 stable cell line
  • Construction and application of PK-15 stable cell line
  • Construction and application of PK-15 stable cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 1

[0025] Embodiment 1 Construction of a PK-15 stable cell line

[0026] 1 Construction of recombinant lentiviral vector

[0027] Consult the porcine CD163 (HM991330) and SBQ (Siglec10, KJ670156) gene sequences reported by GenBank in the National Center for Biotechnology Information (NCBI), use the primer design software Premier 6 and Oligo 7.57 to design the primer sequences of CD163 and SBQ genes, and amplify Bases 1-3348 of the CD163 gene (the entire coding region sequence) and bases 1-1860 of the SBQ gene (the entire coding region sequence). The amplified product of the CD163 gene was digested with Nhe I and BamH I and ligated to the PCDH-CMV-MCS-EF1A-MCHEERY-T2A-PURO vector. The amplified product of the SBQ gene was digested with EcoR I and BamH I and connected to the PLVX-CBA-IRES-NEO-T2A-EGFP vector, and then the two vectors were transformed into DH5a competent cells. Plasmids were extracted for enzyme digestion identification and sequencing. The correctly constructed v...

Embodiment 2

[0038] Example 2PK-15 CD163+SBQ Application of Stable Cell Lines

[0039] 1 Cell Subculture Method

[0040] Take out the PK-15 from the liquid nitrogen tank CD163+SBQ The cell line cell tube was placed in a 37°C water bath to melt rapidly, and the PK-15 CD163+SBQ Cell line F2 was transferred into a centrifuge tube containing 15ml of serum-free medium, and centrifuged at 1000rpm for 5 minutes. Use DMEM medium (containing 8% FBS by volume) to suspend the cells, culture at 37°C, digest the cells with trypsin when the coverage rate reaches 100%, subculture at a ratio of 1:3 to 1:4, and cultivate unmodified PK- 15 cells and Marc-145 cells were used as controls.

[0041] 2PK-15 CD163+SBQ Cultivation of cell lines in bioreactors

[0042] cultured PK-15 CD163+SBQ The cell lines were digested and passaged, and divided into 4 T25 square flasks for culture. PK-15 cells and Marc-145 cells were also divided into four T25 cells, and the cells were inoculated when the cells grew to a...

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Abstract

The invention relates to the technical fields of cytobiology, genetic engineering and veterinary biologicalogy, in particular to a construction method and application of a PK-15 stable cell strain. The construction method of the cell line for porcine reproductive and respiratory syndrome virus (PRRSV) breeding comprises the following steps that 1) a CD163 gene and an SBQ gene are constructed intoa lentiviral vector separately to obtain a recombinant lentiviral vector; 2) the recombinant lentiviral vector and a virus rescue helper plasmid are transferred into a mammalian cell, and the cell iscultured to obtain a recombinant virus; 3) a PK-15 cell is injected with the recombinant virus of step 2) to obtain the cell line. The invention further provides application of the cell. The obtainedcell has stable hereditary characters, is sensitive to the PRRSV, and has a very good application prospect.

Description

technical field [0001] The invention relates to the technical fields of cell biology, genetic engineering and veterinary biological products, in particular to a construction method and application of a stable PK-15 cell line. Background technique [0002] Porcine reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus, PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, PRRS), also known as "blue ear disease". PRRSV has typical monocytic characteristics. In vivo, it mainly infects porcine alveolar macrophages (PAM), and in vitro, it mainly infects African green monkey kidney cell line Marc-145. Therefore, the industry mainly uses Marc-145 as a host to cultivate PRRSV for the production of related vaccines. [0003] However, the propagation of PRRSV in Marc-145 cells has the limitation that the virus content is not high. Therefore, increasing the virus conten...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N5/10C12N7/00C12R1/93
CPCC12N15/86C12N5/0686C12N7/00C12N2740/15043C12N2800/107C12N2510/00C12N2501/599C12N2770/10051
Inventor 邢刚粟硕黄杰贺微岳丰雄徐祥兰王洁清刘原子何洪奎江勇王立斌
Owner 成都史纪生物制药有限公司
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