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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: 2007-08-08
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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Construction of pCDNA3.1(+)-BMP2

[0065] 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, a fragment with a size of about 1200 bp was amplified by polymerase chain reaction ( FIG. 2A ). This fragment was cloned into a eukaryotic expression vector to obtain pCDNA3.1(+)-BMP2, which was confirmed to be consistent with the expected target sequence by restriction enzyme digestion (Fig. 2B) and sequencing.

[0066] B2F: 5'-gctggatccaccatggtggccgggacccgctgtc-3'

[0067] B2R: 5'-gcgtctcgagctagcgacacccacaaccctccac-3'

Embodiment 2

[0068] Example 2: Establishment of engineering cell strains stably and efficiently expressing recombinant human BMP2

[0069]Dihydrofolate reductase-deficient Chinese hamster ovary cells (CHO-dhfr-) use a complete medium supplemented with glycine, hypoxanthine, thymine 10% FBS IMEDM (Hyclone), at 37 ° C, 5% CO2, 10 cm culture plate Culture cells to about 80% confluency. Use cationic liposome Lipofectine 2000 (Invitrogen) to pCDNA3.1(+)-BMP2 and pSV2-DHFR at a ratio of 10:1, transfect according to the method in the operation manual, and then use the selection method containing 700ug / ml G418 and 10% FCS IMDM Media screened neo and dhfr positive clones. The obtained positive clones were divided into 1×10 6 / 10ml cells were inoculated on a 10cm cell culture dish, and the cells were gradually cultured with IMDM medium containing 100nM and 1uM methotrexate (MTX) and 10% FBS to amplify the target gene, and finally obtained a medium containing 1uM MTX Normal growing recombinant cel...

Embodiment 3

[0070] Example 3: Analysis of bone formation activity induced by recombinant human BMP2 in vitro

[0071] When the myoblast cell line C2C12 was cultured to 70-80% confluence with DMEM containing 10% FBS, washed with PBS, digested with trypsin, and counted, 2×10 4 Cells / well were 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 the conditioned medium containing 5%, 10%, and 20% rCHO (BMP2) 24h cell culture solution was added to stimulate the culture of C2C12. After 5 days, count with the CCK-8 kit The number of cells per well, and then the alkaline phosphatase activity was measured by colorimetric method (Figure 4), the highest expression level can reach 7.83 μg / 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 / 24h*106; 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) highly expressing human bone morphogenetic protein 2 and its establishment method. Specifically, an immortalized mammalian cell line that is stably transfected with the coding sequence of the human bone morphogenetic protein 2 (hBMP2) gene is constructed, and a cloning cell line that stably and efficiently expresses rhBMP2 is obtained after pressure selection and gene amplification. Background technique [0002] Due to trauma, infection, tumor and dysplasia, the bone loses some bone quality, and the symptoms such as refractory bone defect, nonunion and delayed fracture union are one of the most difficult problems in clinical practice. The best way to treat bone defects is autologous bone grafting, but in addition to the limited source and the possibility of complications in the bone harvesting area, the repair speed of autol...

Claims

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Application Information

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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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