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Construction and application of Marc-145 stable cell strain

A cell line and cell technology, applied in the fields of genetic engineering, veterinary biological products, and cell biology, can solve the problem of low virus content and achieve the effects of strong genetic stability, good immunogenicity, and high sensitivity

Pending Publication Date: 2019-11-08
成都史纪生物制药有限公司
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  • Summary
  • 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 Marc-145 stable cell strain
  • Construction and application of Marc-145 stable cell strain
  • Construction and application of Marc-145 stable cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Construction of a Marc-145 Stable Cell Line

[0026] 1 Construction of recombinant lentiviral vector

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

Embodiment 2

[0038] Example 2 Marc-145 CD163+SBQ Application of Stable Cell Lines

[0039] 1 Cell Subculture Method

[0040] Take out the Marc-145 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 Marc-145 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 Marc- 145 cells served as a control.

[0041] 2Marc-145 CD163+SBQ Cultivation of cell lines in bioreactors

[0042] The above-mentioned transformed cell Marc-145 CD163+SBQ Proliferation culture was carried out on the cell line Cytodex I. The cell culture parameters are: dissolved oxygen 40%, rotation speed 40r / m, pH value 7.2, culture volume 3L, cell culture me...

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Abstract

The invention relates to the technical fields of cytobiology, genetic engineering and animal biological products, in particular to a construction method and application of a Marc-145 stable cell strain. The invention provides the construction method of the cell strain for porcine reproductive and respiratory syndrome virus (PRRSV) reproduction, the construction method comprises the following stepsthat 1), a CD163 gene and a SBQ gene are correspondingly constructed to an lentiviral vector, and a recombination lentiviral vector is obtained; 2), the recombination lentiviral vector and a virus rescue auxiliary plasmid are together transmitted into a mammalian cell, the cell is cultured to obtain recombination viruses; and 3) the recombination viruses in the steps 2) infect a Marc-145 cell, and the Marc-145 stable cell strain is obtained. The invention further provides use of the cell. An inheritable character of the cell is stable, and the cell is sensitive to PRRSV, and has very good application prospects.

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 Marc-145 cell line. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is the pathogen that causes porcine reproductive and respiratory syndrome (PRRS), also known as "blue ear disease". PRRSV has typical monophilic properties. In vivo, it mainly infects porcine alveolar macrophage (PAM), and in vitro, it mainly infects the 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 proliferation of PRRSV in Marc-145 cells has the limitation of low viral content. Therefore, increasing the viral content of PRRSV propagated in vitro is one of the important ways to improve the quality of related vaccine products...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C07K14/47C12N7/00
CPCC12N15/86C07K14/70596C07K14/47C12N5/0686C12N7/00C12N2740/15043C12N2770/10051C12N2510/00
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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