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目的克隆小鼠内皮抑素(Endostatin)编码区 cDNA 序列及测序鉴定并构建 pEgr-IFNY-Endostatin 双基因表达载体。方法从小鼠肝细胞提取总 RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增获得全长 Endostatin 基因,测序并利用基因重组技术构建 pEgr-IFNY-Endostatin 双基因共表达质粒。结果分光光度计测定50倍稀释的总 RNA 抽提产物,A_(260)=0.834,A_(280)=0.407,A_(260):A_(280)=2.049,总 RNA 浓度为1.668g/L。EndostatincDNA 的RT-PCR 产物与载体 pMD18T 连接并经测序证实 Endostatin 序列与文献报道完全一致,并构建了含 Egr-1启动子的 IFNY 和 Endostain 双基因共表达质粒 pEgY-IFNY-Endostatin。结论成功克隆小鼠 Endostatin 基因,构建了pEgr-IFNY-Endostatin 双基因共表达质粒。
Objective To clone the cDNA sequence of mouse endostatin and identify the recombinant plasmid pEgr-IFNY-Endostatin. Methods Total RNA was extracted from hepatocytes of mice. The full - length Endostatin gene was amplified by reverse transcription - polymerase chain reaction (RT - PCR). The recombinant plasmid pEgr - IFNY - Endostatin was co - expressed by sequencing. Results The total RNA extracted by 50-fold dilution was determined by spectrophotometer. The results showed that A 260 and 0.8 280 were equal to 0.407 and A 260 respectively. The total RNA concentration was 1.668 g / L. EndostatincDNA RT-PCR product was ligated with the vector pMD18T and sequenced to confirm that the sequence of Endostatin was completely consistent with that reported in the literature. The IFNγ and Endostain co-expression plasmid pEgY-IFNY-Endostatin with the Egr-1 promoter was constructed. Conclusion The murine Endostatin gene was successfully cloned and the pEgr-IFNY-Endostatin double gene co-expression plasmid was constructed.