[Expression, characterization and biological activity analysis of recombinant human bone morphogenetic protein 2 in CHO cells].
Sheng Wu Gong Cheng Xue Bao
; 22(6): 968-72, 2006 Nov.
Article
en Zh
| MEDLINE
| ID: mdl-17168321
ABSTRACT
Bone morphogenetic protein 2(BMP-2) is a member of the of BMPs family, its osteoinductive capacity has already been demonstrated. We tried to express hBMP-2 in CHO cell. In this study, we inserted hBMP-2 cDNA into vector pCDNA3.1(+) to construct hBMP-2 eukaryotic expression vector pCDNA3.1(+)-hBMP-2. Recombinant Chinese hamster ovary (rCHO) cell line expressing high-level recombinant human bone morphogenetic protein 2(rhBMP-2) was constructed by co-transfecting the expression vectors pCDNA3.1(+)-hBMP-2 and plasmid pSV2-dhfr into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.1 and 1 micromol/L. Western blot analyses showed a specific band of about 18 kD in reduced sample lane and a specific band of about 32 kD in non-reduced sample lane, this indicated that rCHO cells secret rhBMP-2 as a homodimeric glycoprotein form. Finally, we obtained a single clone cell strain expressing a high level (7.83 microg/24 h/10(6) cells) of rhBMP-2 tested by ELISA. Biological activity of rhBMP-2 was tested by the induction of alkaline phosphatase(ALP) activity in C2C12 cells. We treated C2C12 with different concentration of rhBMP-2 condition medium(CM) for 5d. The results showed that the rhBMP-2 could significantly increase the ALP activity of C2C12.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas Recombinantes
/
Proteína Morfogenética Ósea 2
Límite:
Animals
/
Humans
Idioma:
Zh
Revista:
Sheng Wu Gong Cheng Xue Bao
Asunto de la revista:
BIOTECNOLOGIA
Año:
2006
Tipo del documento:
Article
País de afiliación:
China