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目的构建人源Dickkopf-1(DKK-1)基因的真核表达质粒,为进一步探讨DKK-1的生物学功能奠定基础。方法Trizol法从人新鲜胎盘组织中抽提总RNA,RT-PCR法反转录为cDNA,以cDNA为模板扩增人源DKK-1的基因序列,将真核表达载体pcDNA3.1和DKK-1的PCR产物分别在双酶切后用T4连接酶连接,构建重组质粒pcDNA3.1-DKK-l,转化至大肠杆菌后经菌落PCR、NHeⅠ和EcoRⅠ双酶切及测序鉴定。结果RT-PCR法从人胎盘中扩增出816bp大小的目的片段,重组质粒转化至大肠杆菌后菌落PCR同样扩增出816bp大小的目的片段,NHeⅠ和EcoRⅠ双酶切重组质粒能切出两条目的条带,测序后与Genbank中DKK-1的cDNA对比,证明DKK-1的基因序列完全正确。结论成功构建了pcDNA3.1-DKK-1重组质粒,为研究人源DKK-1的功能及其在细胞系中的作用奠定了基础。
Objective To construct eukaryotic expression plasmid of human Dickkopf-1 (DKK-1) gene and lay a foundation for further study on the biological function of DKK-1. Methods Trizol method was used to extract total RNA from human fresh placenta tissue and RT-PCR was reverse transcribed into cDNA. CDNA was used as a template to amplify the human DKK-1 gene sequence. The eukaryotic expression vectors pcDNA3.1 and DKK- 1 PCR products were double digested with T4 ligase to construct recombinant plasmid pcDNA3.1-DKK-l, transformed into E. coli after colonies PCR, NHe Ⅰ and EcoR Ⅰ double digestion and sequencing identified. Results The target fragment of 816bp was amplified by RT-PCR from human placenta. After the recombinant plasmid was transformed into E. coli, the target fragment of 816bp was amplified by PCR. Two recombinant plasmids were digested by NHeⅠ and EcoRⅠ. The DNA sequence of DKK-1 was completely correct after sequencing compared with the cDNA of DKK-1 in Genbank. Conclusion The pcDNA3.1-DKK-1 recombinant plasmid was successfully constructed, which laid the foundation for studying the function of human DKK-1 and its role in cell lines.