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Rat bone marrow tumour cell NSO and preparation method and application thereof

A technique for myeloma cells and mouse myeloma, which is applied in the field of mouse myeloma cell NSO and its preparation, can solve the problems of increasing infection probability, difficulty in purification, and increasing purification cost, avoiding animal-derived components and unsafe factors, reducing Purification cost and loss, the effect of high culture efficiency

Inactive Publication Date: 2012-05-02
北京普赛资产管理有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the addition of lipids will introduce animal-derived components, increasing the chance of infection and the difficulty of purification, which will also increase the cost of purification

Method used

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  • Rat bone marrow tumour cell NSO and preparation method and application thereof
  • Rat bone marrow tumour cell NSO and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, prepare the mouse myeloma cell NSO that can grow in serum-free, lipid-free medium

[0047] 1. Preparation

[0048] Mouse myeloma cells NSO cells were purchased from ECACC (The European Collection of Animal Cell Culture).

[0049] (1) Cells were placed in cell culture medium I, and cultured at 37°C for 3 days; the composition of cell culture medium I was: RPMI1640 medium containing 10% (volume percentage) fetal bovine serum (Table 1), The pH is 7.4.

[0050] (2) Carry out a subculture of cells: cells are placed in cell culture medium II, and cultivated at 37° C. for 3 days; Culture medium I), adding an equal volume of hybridoma serum-free medium (Invitrogen Company Catalog Number: 11279-023) containing 1% (volume percentage) bovine serum albumin (BSA) with a pH of 7.4 to allow cell culture The final concentration of fetal bovine serum in Base II was reduced to 5%.

[0051] (3) Carry out secondary cell subculture: cells are placed in cell culture medium ...

Embodiment 2

[0062] Example 2. Production of Human TNF-alpha Soluble Receptor Using Mouse Myeloma Cell NSO-Ch1 CGMCC No.2127

[0063] The cDNA sequence encoding the extracellular segment of the human TNF-alpha type II receptor and the cDNA sequence encoding the Fc segment of human immunoglobulin IgG1 were constructed by PCR into a recombinant gene (TNFR-Fc) (shown in sequence 1 in the sequence listing); TNFR-Fc is inserted into the Xba1 and Xho1 sites of the expression vector pCI-gpt with a selectable marker (guanine phosphoribosyltransferase, gpt) and a gene expression regulatory region (CMV promoter, terminator), to obtain the expression of the recombinant protein Vector pCI-gpt-TNFR-Fc. Linearize pCI-gpt-TNFR-Fc with restriction endonuclease FspI, and introduce it into NSO-Ch1 (CGMCC No.2127) cells by electroporation. The transfected cells were screened with Mycophenolate in medium containing Xanthine to obtain stably transfected cell lines.

[0064] The successfully transfected cells...

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Abstract

The invention discloses a rat bone marrow tumour cell NSO and a preparation method and an application thereof. The rat bone marrow tumour cell NSO can grow in a serum-free and fat-free culture substrate, the preservation number of one strain (NSO-Ch1) is CGMCC No.2127. If the rat bone marrow tumour cell NSO of the invention, which can grow in a serum-free and fat-free culture substrate, is used to express foreign protein, cell culture efficiency and expression efficiency can be ensured, and other animal source ingredients and unsafe factors can be avoided; thus, the invention can effectively lower purification cost and loss and improve the yield and profitability of biological products.

Description

technical field [0001] The invention relates to a mouse myeloma cell NSO and its preparation method and application. Background technique [0002] Large-scale fermentation of mammalian cells is the basis for mass production of antibody drugs for clinical use, and it is also the most critical technology in the field of modern biopharmaceuticals. About 70% of the 31 biotechnology drugs approved by the U.S. Food and Drug Administration for marketing from January 2000 to December 2004 are recombinant proteins expressed by mammalian cells. At present, three cell lines such as CHO, SP2 / 0 and NSO are mainly used in the world to produce recombinant protein therapeutic drugs. For example, Rituximab for the treatment of lymphoma, Infliximab for the treatment of rheumatoid arthritis, and Daclizumab for the prevention of acute organ rejection are expressed with CHO, NSO and SP2 / 0, respectively. [0003] At present, there are very few drug products expressed by mammalian cells approved...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12P21/02
Inventor 朱卫彬唐捷贾俊英李娜
Owner 北京普赛资产管理有限责任公司
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