重组人色素上皮衍生因子在真核细胞SP2／0中的瞬时表达及活性鉴定

Transient expression and biological activity identification of human pigment epithelium-derived factor in mammary cell line SP2/0

目的构建抑制早产儿视网膜病新生血管形成的重组人色素上皮衍生因子（pigment epithelium—derived factor，PEDF）真核表达载体，检测其在小鼠骨髓瘤细胞SP2／0中的瞬时表达。方法根据已知的GenBank中人PEDFcDNA成熟蛋白编码区序列设计特异性引物，以全基因合成的pUC57-PEDF质粒为模板，经聚合酶链反应技术扩增人PEDF基因，定向克隆至真核表达载体pIRESneo3中，获得重组表达质粒pIRESneo3-PEDF。将重组质粒pIRESneo3-PEDF与脂质体转染试剂Lipofectamine^TM 2000混合后转染小鼠骨髓瘤细胞SP2／0，采用酶联免疫吸附试验及Western印迹对表达产物进行鉴定。收集转染细胞培养液上清，采用四甲基偶氮唑盐比色法检测重组PEDF活性。结果聚合酶链反应技术、酶切鉴定和测序结果表明，pIRESneo3PEDF表达载体构建成功。脂质体法将表达质粒成功转染到SP2／0中，经培养，可分泌表达PEDF，且经Western印迹检测证明为人PEDF，分子量为50000。转染36h上清液中PEDF浓度最高，为（0．92±0．04）ug／ml。转染36h细胞培养液上清对人脐静脉内皮细胞的抑制作用最强（P〈0．05）。结论成功构建人PEDF真核表达质粒pIRESneo3PEDF，转染细胞可瞬时分泌表达有活性的人PEDF，对人脐静脉内皮细胞增殖有抑制作用，为开展下一步稳定转染表达及蛋白纯化奠定基础，为早产儿视网膜病的防治带来新的希望。

Objective To construct eukaryotic expression plasmid plRESneo3 pigment epithelium-derived factor （PEDF） and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector plRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3 （4,5-dimethylthiazol-z-yl） 2, 5-diphenytetrazolium bromide （MTT） method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h E（0. 92±0. 04） ug/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h （P〈0.05）. Conclusions The eukaryotic expression plasmid plRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity.