人源Dickkopf-1真核表达质粒的构建

Construction of Recombinant Plasmid pcDNA3.1-Dickkopf-1

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 the recombinant plasmid pc DNA3.1-Dickkopf-1（DKK-1） which is capable of expressing in mammalian cells for further study. Methods DKK-1 gene containing NHe I and EcoR I endoenzyme sites were obtained by using RT-PCR. Double enzyme digestion was conducted for pcDNA3.1 and RT-PCR product of DKK-1 gene.Both fragments were connected by using T4 ligase and transferred to DHSa.Then the connected product was detected by PCR, NHe I and EcoR I double enzyme digestion and suquencing. Results One 816 bp fragment was amplified from human placenta by RT-PCR.The same size fragment was amplified from colony by PCR .Two corresponding fragments were obtained when the recombinant plasmid was digested by NHe Ⅰand EcoR Ⅰ. The result of sequencing was completely correct. Conclusion The recombinant plasmid pcDNA3.1-DKK-1 is succesfully constructed, so as to facilitate the study of the function of DKK-1 in carcinoma cell lines.