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目的:克隆人转化生长因子-β1(TGF-β1)基因编码区cDNA序列,构建并鉴定TGF-β1真核表达载体。方法:设计含有EcoRⅠ和XholⅠ酶切位点的TGF-β1cDNA引物,利用逆转录聚合酶链反应(RT-PCR)法,以人白细胞mRNA为模版,扩增TGF-β1基因,纯化PCR产物,TA克隆,双酶切与pcDNA3质粒表达载体连接,转化感受态大肠杆菌JM109,酶切鉴定阳性重组子,并进行序列测定。结果:琼脂糖凝胶电泳显示,用双酶切后形成分子量1.2kb的条带,符合物理图谱,表明表达载体构建成功,序列测定结果与预测结果完全一致。结论:成功克隆了TGF-β1基因,并成功地构建了真核表达载体pcDNA3-TGF-β1。
OBJECTIVE: To clone the cDNA sequence encoding the transforming growth factor-β1 (TGF-β1) gene and to construct and identify the eukaryotic expression vector of TGF-β1. Methods: TGF-β1 cDNA primers containing EcoRⅠand XholⅠ restriction sites were designed and used to amplify TGF-β1 gene by reverse transcription-polymerase chain reaction (RT-PCR) Cloned and double-digested with pcDNA3 plasmid expression vector, transformed into competent E. coli JM109, digested positive recombinant identified and sequenced. Results: The agarose gel electrophoresis showed that the band of 1.2kb molecular weight was formed by double enzyme digestion, which was in accordance with the physical map, indicating that the expression vector was successfully constructed. The results of sequence analysis were in good agreement with the prediction results. Conclusion: The TGF-β1 gene was successfully cloned and the eukaryotic expression vector pcDNA3-TGF-β1 was successfully constructed.