骨保护素基因修饰的pSecTag2/B载体的构建

The construction of pSecTag2/B vector modified by osteoprotegerin

目的:克隆骨保护素基因(osteoprotegerin,OPG)的cDNA,构建pSecTag2/B-OPG真核分泌表达穿梭载体,为组织工程和基因工程联合治疗牙周病提供物质基础。方法:提取人胚胎肾293细胞的总RNA,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pSecTag2/B载体中,测序鉴定。结果:从293细胞的总RNA中反转录扩增得到编码骨保护素的基因片段,重组到pSecTag2/B载体。经测序证实,此基因与GenBank中[gi:33878056]提供的序列完全一致;重组质粒经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。结论:本研究成功构建了pSecTag2/B-OPG分泌表达穿梭载体。

PURPOSE:In order to treat periodontitis by using tissue engineering and gene engineering technology, we constructed mammalian secreted expression pSecTag2/B-OPG vector,the cDNA sequence encoding osteoprotegerin(OPG) obtaining from 293 cell. METHODS: The primers were designed based on the human OPG cDNA sequence. Total mRNA was isolated from 293 cells and RT-PCR was performed .The fragment was recombined into pSecTag2/B vector and sequenced by automatic sequence analyzer. RESULTS: The sequences of OPG cDNA from 293 cell by RT-PCR were completely identical to the sequences provided by GenBank [gi:33878056]. After polymerase chain reaction and the recombinant plasmid digesting with Hind III,EcoR I and BamH I,1% agarose electrophoresis showed several fragments, which were consistent with predicted size. CONCLUSION: From cultured 293 cells the cDNA has been cloned and pSecTag2/B-OPG vector has been constructed successfully.Supported by Research Fund (No.03043304) from Anhui Provincial Natural Science Foundation.