摘要
目的:克隆人色素上皮衍生因子(PigmentEpithelium-DerivedFactor,PEDF)全长cDNA,建立稳定表达人PEDF分子的哺乳动物细胞系。方法:RT-PCR获取人PEDFcDNA,序列测定后克隆入真核表达载体pCDNA3.0中,用脂质体转染入HepG2细胞,G418筛选出稳定表达细胞系,用RT-PCR与Westernblot分别检测RNA和蛋白表达水平。结果:RT-PCR扩增得到预期大小的目的条带,序列分析表明成熟肽编码区与发表序列完全一致;克隆入pCDNA3.0中得到PEDF真核表达载体,G418筛选3周后得到稳定表达细胞系,RT-PCR与Westernblot显示PEDF在HepG2细胞中有高水平表达。结论:人PEDF全长cDNA成功克隆,并在哺乳动物细胞系HepG2中获得稳定表达,为进一步研究PEDF分子对增生性瘢痕血管形成的作用奠定了理论基础。
AIM:To clone the full length cDNA of human pigment epithelium derived factor(PEDF),and to establish the mammal cell line which can express the PEDF molecule stably. METHODS:Human PEDF cDNA was obtained by RT PCR,and cloned into pCDNA3.0 after confirmed by sequence analysis.HepG2 cells were transfected by LipofectamineTM reagent and then the stable cell line was selected with G418.The expression level of RNA and albumen were detected by RT PCR and Western blot. RESULTS:Purpose strap with expected length was obtained by RT PCR amplification,and the published sequence was matched with encoding mature peptide completely as shown by sequence analysis.Eukaryotic vector expressing PEDF was got by gene cloning,and cells expressing PEDF stably were got by screening with G418 3 weeks after transfection.RT PCR and Western blot showed high level expression of PEDF in HepG2 cells. CONCLUSION:Full length cDNA of human PEDF gene is cloned successfully and stably expressed in HepG2 of the mammal cell line,which provides a theoretical basis for the study of the effects of PEDF on hypertrophic scar angiogenesis.
出处
《中国临床康复》
CSCD
2004年第17期3298-3299,i004,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金资助课题(30271346)~~
