Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E. Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site or the expr...Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E. Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site or the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5a. The proteins or mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was success- fully constructed. By temperature inducing, under the control of the bacteriophage λPL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size or expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control. Conclusion Tke mBDNF gene can be expressed bioactively in E. Coli.展开更多
Brain-derived neurotrophic factor (BDNF) has been considered a new angiogenesis mediator. ProBDNF, the precursor of BDNF, plays opposite neuronal functions to BDNF, but the role of proBDNF on angiogenesis remains unkn...Brain-derived neurotrophic factor (BDNF) has been considered a new angiogenesis mediator. ProBDNF, the precursor of BDNF, plays opposite neuronal functions to BDNF, but the role of proBDNF on angiogenesis remains unknown. We found human umbilical vein endothelial cells (HUVEC) expressing BDNF, proBDNF, p75<sup>NTR</sup>, Sortilin and TrkB. ProBDNF significantly decreased HUVEC viability in MTT assay, and this inhibition was neutralized by anti-proBDNF. Endothelial cell tube formation assay showed that proBDNF significantly inhibits HUVEC angiogenesis in vitro. Matrigel plug assay disclosed that proBDNF also impeded angiogenesis in vivo, while anti-proBDNF greatly facilitated angiogenesis. Immunostaining of CD31 and α-SMA in Matrigel plugs confirmed the inhibitive effect of proBDNF on angiogenesis. In conclusion, proBDNF can act as an angiogenesis inhibitor. It added more evidence to the “Yin-Yang” theory by showing mBDNF is a mediator of angiogenesis as “Yang” and proBDNF works as an angiogenesis inhibitor as “Yin”.展开更多
文摘Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E. Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site or the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5a. The proteins or mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was success- fully constructed. By temperature inducing, under the control of the bacteriophage λPL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size or expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control. Conclusion Tke mBDNF gene can be expressed bioactively in E. Coli.
文摘Brain-derived neurotrophic factor (BDNF) has been considered a new angiogenesis mediator. ProBDNF, the precursor of BDNF, plays opposite neuronal functions to BDNF, but the role of proBDNF on angiogenesis remains unknown. We found human umbilical vein endothelial cells (HUVEC) expressing BDNF, proBDNF, p75<sup>NTR</sup>, Sortilin and TrkB. ProBDNF significantly decreased HUVEC viability in MTT assay, and this inhibition was neutralized by anti-proBDNF. Endothelial cell tube formation assay showed that proBDNF significantly inhibits HUVEC angiogenesis in vitro. Matrigel plug assay disclosed that proBDNF also impeded angiogenesis in vivo, while anti-proBDNF greatly facilitated angiogenesis. Immunostaining of CD31 and α-SMA in Matrigel plugs confirmed the inhibitive effect of proBDNF on angiogenesis. In conclusion, proBDNF can act as an angiogenesis inhibitor. It added more evidence to the “Yin-Yang” theory by showing mBDNF is a mediator of angiogenesis as “Yang” and proBDNF works as an angiogenesis inhibitor as “Yin”.
