神经生长因子基因的克隆及在昆虫细胞中的表达

Cloning and Expression of Gene Encoding Human NGF in Insect Cells

以人胎盘组织DNA为模板,经PCR扩增出包含信号肽,前肽和成熟肽部分的prepro-NGF序列,经序列分析后克隆至转移载体pFastBac1中得到pFastBac-NGF,再将其转化含穿梭载体Bacmid的大肠杆菌DH10Bac,发生转座作用后,得到含NGF基因的重组穿梭载体rBacmid-NGF,用纯化的rBacmid-NGFDNA直接转染培养的昆虫细胞Sf9,得到重组病毒rAcV-Bac-hGH,经PCR和酶切鉴定,NGF基因正确地插入在病毒基因组的多角体蛋白基因启动子下.表达产物经SDS-PAGE和Western blot检测在34 kD左右有一表达带并具有神经生长因子的免疫学活性.

Nerve growth factor is one of the most important factors,which play an important role in regulating the growth,development and survival of the neuron.The purified NGF from human placenta has been reported,the tissue from which the NGF can be isolated is very limited.It is important for basic research and clinic application to express hNGF by genetic engineering. Here the NGF gene encoding the pre-pro-NGF was amplified using the DNA of human placenta as template.The amplified fragment was sequenced and subcloned into the baculovirus transfer vector pFastBac1,the constructed recombinant plasmid pFastBacNGF was transferred into E.coli DH10Bac which includes a shuttle vector,Bacmid.By site-specific transposition,NGF gene was integrated into Bacmid,and a recombinant shuttle vector was constructed,named rBacmid-NGF.The cultured Sf9 cells were directly transfected with the rBacmid-NGF DNA,and the pure recombinant baculovirus rAcV-Bac-NGF was obtained.PCR and restriction enzyme assay revealed that NGF gene was correctly inserted into baculovirus genome under the control of polyhedrin promoter.SDS-PAGE and Western blot assay revealed that the expressed NGF in Sf9 cells had a Mr 34 kD and displayed immunological activity of NGF.