广西眼镜蛇蛇毒神经生长因子基因在NIH3T3细胞中的表达

EXPRESSION OF NERVE GROWTH FACTOR GENE FROM NAJA NAJA ATRA IN NIH3T3 CELL LINE

目的:构建广西眼镜蛇蛇毒神经生长因子(NGF)基因的真核表达载体pcDNA3.1(-)-NGF,并在NIH3T3细胞中进行表达。方法:采用RT-PCR的方法扩增蛇毒NGF的基因片段,将目的基因、真核表达载体pcDNA3.1(-)分别酶切后回收,连接、转化而构建重组表达载体pcDNA3.1(-)-NGF,经酶切和测序对重组体进行鉴定。利用脂质体Lipofectamine～(TM) 2000方法转染NIH3T3细胞,对阳性克隆裂解上清中NGF的表达用SDS-PAGE、Western-blot的方法检测。结果:重组载体经酶切、测序分析,插入的基因片段为表达蛇毒NGF的基因,NGF基因在NIH3T3细胞中获得表达。结论:成功构建了真核表达载体pcDNA3.1(-)-NGF,并检测出在哺乳动物细胞NIH3T3中表达,为进一步开展NGF基因治疗神经系统的疾病奠定了实验基础。

Objective:To construct the eukaryotic expression vector pcDNA3.1(-)-NGF of nerve growth factor gene from Naja naja atra(Chinese cobra) and express it in NIH3T3 cells.Methods:The gene of Cobra NGF was amplified by RT-PCR.The RT-PCR products and the expression vector pcDNA3.1(-) were digested respectively,then reclaimed,ligated,transformed to construct recombinant expression vector pcDNA3.1(-)-NGF.The recombinant plasmid was identified by the restricted enzymes and the sequence analysis.NIH3T3 cell was transfected with this vector using LipofectamineTM2000 transfection reagent.The expression of NGF in lysate of positive clones was analyzed by SDS-PAGE and Western-blot.Results:Through digesting and sequencing,the insert DNA was the gene which expressed the protein of cobra NGF.The NGF gene was expressed successfully in NIH3T3 cells.Conclusion:The eukaryotic expression vector pcDNA3.1(-)-NGF was constructed and the NGF gene was expressed in the transfected NIH3T3 cells successfully,which provided the basis for gene therapy of nervous system diseases.