mGM-CSF重组质粒的构建、表达及活性鉴定

Construction,Expression and its Activity Examinations of Recombiant Plasmid with mGM-CSF

目的 构建pc mGM CSF重组质粒载体 ,为mGM CSF基因治疗肿瘤的研究奠定基础。方法 采用RT PCR方法从小鼠脾脏中获得目的基因mGM CSF ,克隆于 pcDNA3 .1/Myc His( -) (A )质粒上 ,成为pc mGM CSF ,用PCR、酶切进行鉴定 ,然后用脂质体转染小鼠骨髓瘤细胞SP2 / 0 ,用G 418筛选后通过RT PCR和SDS PAGE鉴定 ,将转染SP2 / 0上清加入NFS 60细胞 ,检测蛋白活性。结果 重组质粒中含有mGM CSF基因 ,在SP2 / 0中有表达 ,且表达产物能分泌到肿瘤细胞外 ,分泌到细胞外的产物用mGM CSF依赖株NFS 60细胞检测证明具有生物学活性。结论 成功构建含mGM CSF真核表达重组质粒 。

Objective To construct recombiant plasmid pc-mGM-CSF and lay a primary foundation for further study of mGM-CSF gene therapy for tumors.Methods mGM-CSF obtained from mouse spleen cells by RT-PCR was cloned into pcDNA 3.1/Myc-His(-)(A)to form pc-mGM-CSF which was transfected into SP2/0 via lipsome after identification of PCR,restriction enzyme digesting and DNA sequencing.The SP2/0 selected by G418 was identified by RT-PCR and SDS-PAGE.NFS-60 cells was added into the supernatant of the cells to examine the activity of the protein.Results The recombiant plasmid had mGM-CSF gene which was expressed in SP2/0 in vitro and the expressed protein was secreted out of the cells.The secreted protein sustained the growth of NFS-60 whose growth was depended on GM-CSF,indicating that mGM-CSF had a biologically activity.Conclusion An eukaryotic expression recombinant plasmid pc-mGM-CSF can be constructed successfully which provides the possibility of further researches on its anti-tumor effect.