CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof

A high-efficiency expression, cell line technology, applied in the field of genetic engineering pharmaceuticals, can solve the problem that the recombinant CHO cell line has not yet been reported.

Inactive Publication Date: 2009-12-09
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] At present, the search results of relevant Chinese patent data show that by co-transfecting human BMP2 and DHFR genes into DHFR-deleted CHO cells, after screening and amplification, recombinant CHO cell lines that express human BMP2 efficiently and stably have not been reported yet.

Method used

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  • CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof
  • CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof
  • CHO cell strain for highly effective expressing rhBMP2 and establishing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Construction of pCDNA3.1(+)-BMP2

[0066] According to the gene sequence of human bone morphogenetic protein 2 (BMP2, SEQ ID NO. 1), two pairs of specific primers (B2F, B2R) were designed and synthesized. Using B2F and B2R as primers, polymerase chain reaction was used to amplify a fragment with a size of about 1200bp ( figure 2 A). This fragment was cloned into a eukaryotic expression vector to obtain pCDNA3.1(+)-BMP2, which was digested ( figure 2 B). Sequencing confirmed that it was consistent with the expected target sequence.

[0067] B2F: 5’-gctggatccaccatggtggccgggacccgctgtc-3’

[0068] B2R: 5’-gcgtctcgagctagcgacacccacaaccctccac-3’

Embodiment 2

[0069] Example 2: Establishment of an engineered cell line stably and efficiently expressing recombinant human BMP2

[0070] Dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr-) with a complete medium supplemented with glycine, hypoxanthine, thymine 10% FBS IMEDM (Hyclone), at 37℃, 5% CO2, 10cm culture plate Culture the cells to about 80% confluence. Use cationic liposome Lipofectine 2000 (Invitrogen) to transfect pCDNA3.1(+)-BMP2 and pSV2-DHFR at a ratio of 10:1 according to the operation manual, and then use the selection of IMDM containing 700ug / ml G418 and 10% FCS The medium was used to screen neo and dhfr positive clones. The positive clones obtained are 1×10 6 / 10ml cells were inoculated in a 10cm cell culture dish, and the cells were gradually cultured in IMDM medium containing 100nM and 1uM methotrexate (MTX) and 10% FBS to amplify the target gene, and finally obtain a medium containing 1uM MTX Recombinant cells that grow normally. Extract the cell cultu...

Embodiment 3

[0071] Example 3: In vitro bone formation activity analysis of recombinant human BMP2

[0072] When the myoblast cell line C2C12 was cultured in DMEM containing 10% FBS to 70-80% confluence, washed with PBS, trypsinized, and counted to 2x10 4 Each cell / well was seeded in a 48-well cell culture plate and incubated in a 37°C, 5% CO2 incubator. After 24 hours, the medium was aspirated, washed twice with PBS, and conditioned medium containing 5%, 10%, and 20% rCHO(BMP2) 24h cell culture medium was added to stimulate the culture of C2C12. After 5 days, count with CCK-8 kit The number of cells per well, and then the colorimetric method to measure the alkaline phosphatase activity ( Figure 4 ), the highest expression level can reach 7.83μg / 24h / 10 6 cell.

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Abstract

The invention discloses a CHO cellar strain of high-effective expressive exogenous rhBMP2, which is characterized by the following: adopting CHO-dhfr- in the dihydrofolic acid reductase flaw-typed Chinese hamster ovary cell as host cell; infecting exogenous hBMP2 plasmid; adopting secretory expressive product to induce myoblast system C2C12 to display the activity to stimulate sclerotomal cell; obtaining single-clone strain of CHO (rhBMP-2) with the content of BMP2 at 7.83ug / 24híñ106; fitting for clinic treatment.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering pharmacy, and relates to a recombinant CHO cell CHO (rhBMP-2) that efficiently expresses human bone morphogenetic protein 2 (rhBMP-2) and a method for establishing it. Specifically, an immortalized mammalian cell line stably transfected with the coding sequence of the human bone morphogenetic protein 2 (hBMP2) gene was constructed. After pressure screening and gene amplification, a clonal cell line stably and efficiently expressing rhBMP2 was obtained. Background technique [0002] Due to trauma, infection, tumors, and developmental abnormalities, the bone loses some bone mass. The formation of refractory bone defects, nonunion, and delayed fracture healing are one of the thorny problems in clinical practice. The best way to treat bone defects is autologous bone transplantation. However, in addition to the limited sources and the possibility of complications in the bone removal area, the re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/12A61K35/12C12N15/09A61K38/18A61K48/00
Inventor 朱天慧张道永闫继东杨爽
Owner NANKAI UNIV
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