【摘要】 目的:构建pSecTag opg真核分泌表达穿梭载体,培养经opg基因修饰的Cos 7细胞系并检测其表达能力,为基因工程技术治疗牙周病提供物质基础。方法:提取293细胞的总mRNA,设计和合成特异性引物,进行逆转录-聚合酶链反应(RT PCR),扩增出具有抑制破骨细胞功能的opg基因cDNA,并将其重组到载体中测序;用脂质体法将目的基因转染至Cos 7细胞,应用免疫组织化学和Westernblot方法证实转染的细胞表达OPG。结果:重组质粒pSecTag2 /B opg经PCR、HindIII单酶酶切及EcoRI和BamHI双酶酶切,电泳显示均能切下与预期大小相符的片段,经测序证实此基因与GenBank中[gi: 33878056]提供的序列完全一致;经免疫组织化学和Westernblot方法检测,在转染opg基因的Cos 7细胞中有OPG表达。结论:成功构建了pSecTag2 /B opg分泌表达穿梭载体,并培养出高分泌表达OPG的Cos 7细胞系。更多还原

【Abstract】 Objective:To transfect human osteoprotegerin( o pg) gene into Cos-7 cells.Methods:The primers were designed b ased on the human opg cDNA sequence,total mRNA was isolated from 293 cells and RT-PCR was performed.The fragment of opg cDNA was inserted into pSecTag 2/B vector and sequenced by automatic sequence analyzer. The recombined plasmid was transfected into Cos-7 cells by Lipofectamine 2000 and OPG protein expressi on in Cos-7 cells was determined by immunohistochemistry and Western blot. Results:The sequence of opg cDNA from 293 cells obtained by RT- PCR was identical to the sequence provided by GenBank [gi:33878056]. The fragm ents of the recombinant plasmid digested with Hind Ⅲ,EcoR I and BamH Ⅰ and exa mined by 10 g/L agarose electrophoresis were consistent with predicted size.OPG over-expressing Cos-7 cells was selected and confirmed by immunohistochemistry and Western blot.Conclusion:Human opg gene may be transfect ed into Cos-7 cells.更多还原