AIM: To obtain nerve growth factor subunit ?(NGF ?) gene from Chinese fetal hippocampus tissue, prove its sequence, clone the mature peptide sequence, and make it express in E.coli .METHODS: Total RNA was extracted, amplified by RT-PCR method. Its 650 bp DNA sequence was inserted into PCR Ⅱ vector. PCR/NGF ? vector was used as the template to amplfy the C-terminate mature peptide sequence, then subcloned it into PG5 vector. The recombinant was transferred into E.coli BL21. BL21 was cultured and induced by IPTG. The activity of the expressed product was measured after purified and refolded.RESULTS: A complete cDNA sequence was determined as 1 047 nucleotides. The cloned 636 bp encoding 212 amino acids was proved homological to Genbank by sequence analysis. The expressed mature peptide showed an clear band of the prospected 14 kD by SDS-PAGE electrophoresis, and was testified by Western blot. The expression level was about 10% of the total cell lysate. CONCLUSION: The chinese NGF ? gene was homological to the foreigners'. The recombinant NGF ? was efficiently expressed in E.Coli and the recombinant protein has high immunological activities.

BACKGROUND:Vocuolar-type ATPase (V-ATPase) is highly expressed in osteoclasts and especial y plays an important role in osteoclastic bone resorption. Tumor necrosis factor-αis a potent stimulator of bone resorption. However, the effect of tumor necrosis factor-αon expression and activity of V-ATPase is stil not clear.
OBJECTIVE:To investigate the mechanism of tumor necrosis factor-αto promote osteoclastic bone resorption by observing expression and activity of V-ATPase.
METHODOsteoclasts cultured in vitro were intervened by different concentrations of tumor necrosis factor-α(5, 10, 30μg/L) in order to observe the changes in expression and enzyme activity of V-ATPase and its effects on bone resorption of osteoclasts. Under an inverted microscope, we observed the formation of resorption lacunas, and bone resorption area was analyzed using Image J software.
RESULTS AND CONCLUSION:The expression and activity of V-ATPase increased significantly after 48 hours of tumor necrosis factor-αintervention and the increase of tumor necrosis factor-αconcentration might enhance this effect. In addition, osteoclastic bone resorption was promoted after intervention with tumor necrosis factor-α. The bone-resorbing capabilities of osteoclasts increased in paral el with the concentration of tumor necrosis factor-α.The results suggested that tumor necrosis factor-α, as a significant inflammatory mediator involved in the pathological process of bone resorption, not only promotes formation of osteoclasts but also enhances bone-resorbing capabilities of osteoclasts by increasing V-ATPase expression and activity.