中华围产医学杂志
中華圍產醫學雜誌
중화위산의학잡지
CHINESE JOURNAL OF PERINATAL MEDICINE
2011年
6期
347-353
,共7页
戴仪%石文静%陈超%王羽雄%于敏
戴儀%石文靜%陳超%王羽雄%于敏
대의%석문정%진초%왕우웅%우민
视网膜病,早产儿%眼蛋白质%神经生长因子类%舍平类%真核细胞
視網膜病,早產兒%眼蛋白質%神經生長因子類%捨平類%真覈細胞
시망막병,조산인%안단백질%신경생장인자류%사평류%진핵세포
Retinopathy of prematurity%Eye proteins%Nerve growth factors%Serpins%Eukaryotic cells
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PEDF cDNA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LipofectamineTM 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,pIRESneo3-PEDF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50 000.转染36 h上清液中PEDF浓度最高,为(0.92±0.04) μg/ml.转染36 h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pIRESneo3-PEDF,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.
目的 構建抑製早產兒視網膜病新生血管形成的重組人色素上皮衍生因子(pigment epithelium-derived factor,PEDF)真覈錶達載體,檢測其在小鼠骨髓瘤細胞SP2/0中的瞬時錶達.方法 根據已知的GenBank中人PEDF cDNA成熟蛋白編碼區序列設計特異性引物,以全基因閤成的pUC57-PEDF質粒為模闆,經聚閤酶鏈反應技術擴增人PEDF基因,定嚮剋隆至真覈錶達載體pIRESneo3中,穫得重組錶達質粒pIRESneo3-PEDF.將重組質粒pIRESneo3-PEDF與脂質體轉染試劑LipofectamineTM 2000混閤後轉染小鼠骨髓瘤細胞SP2/0,採用酶聯免疫吸附試驗及Western印跡對錶達產物進行鑒定.收集轉染細胞培養液上清,採用四甲基偶氮唑鹽比色法檢測重組PEDF活性.結果 聚閤酶鏈反應技術、酶切鑒定和測序結果錶明,pIRESneo3-PEDF錶達載體構建成功.脂質體法將錶達質粒成功轉染到SP2/0中,經培養,可分泌錶達PEDF,且經Western印跡檢測證明為人PEDF,分子量為50 000.轉染36 h上清液中PEDF濃度最高,為(0.92±0.04) μg/ml.轉染36 h細胞培養液上清對人臍靜脈內皮細胞的抑製作用最彊(P<0.05).結論 成功構建人PEDF真覈錶達質粒pIRESneo3-PEDF,轉染細胞可瞬時分泌錶達有活性的人PEDF,對人臍靜脈內皮細胞增殖有抑製作用,為開展下一步穩定轉染錶達及蛋白純化奠定基礎,為早產兒視網膜病的防治帶來新的希望.
목적 구건억제조산인시망막병신생혈관형성적중조인색소상피연생인자(pigment epithelium-derived factor,PEDF)진핵표체재체,검측기재소서골수류세포SP2/0중적순시표체.방법 근거이지적GenBank중인PEDF cDNA성숙단백편마구서렬설계특이성인물,이전기인합성적pUC57-PEDF질립위모판,경취합매련반응기술확증인PEDF기인,정향극륭지진핵표체재체pIRESneo3중,획득중조표체질립pIRESneo3-PEDF.장중조질립pIRESneo3-PEDF여지질체전염시제LipofectamineTM 2000혼합후전염소서골수류세포SP2/0,채용매련면역흡부시험급Western인적대표체산물진행감정.수집전염세포배양액상청,채용사갑기우담서염비색법검측중조PEDF활성.결과 취합매련반응기술、매절감정화측서결과표명,pIRESneo3-PEDF표체재체구건성공.지질체법장표체질립성공전염도SP2/0중,경배양,가분비표체PEDF,차경Western인적검측증명위인PEDF,분자량위50 000.전염36 h상청액중PEDF농도최고,위(0.92±0.04) μg/ml.전염36 h세포배양액상청대인제정맥내피세포적억제작용최강(P<0.05).결론 성공구건인PEDF진핵표체질립pIRESneo3-PEDF,전염세포가순시분비표체유활성적인PEDF,대인제정맥내피세포증식유억제작용,위개전하일보은정전염표체급단백순화전정기출,위조산인시망막병적방치대래신적희망.
Objective To construct eukaryotic expression plasmid pIRESneo3-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 pIRESneo3. 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-y1)-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 [(0.92±0.04) μg/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 pIRESneo3-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.
