[Expression, characterization and biological activity analysis of recombinant human bone morphogenetic protein 2 in CHO cells].
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.
Zhang
Dao-Yong
DY
Laboratory of Medical Molecular Genetics, School of Medicine, Nankai University, Tianjin 300071, China.
Yang
Shuang
S
Lü
Shu-Jun
SJ
Yan
Ji-Dong
JD
Zhu
Tian-Hui
TH
D000469:Alkaline Phosphatase / Q000096:biosynthesis; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2 / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D012995:Solubility
D055396:Bone Morphogenetic Protein 2; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase