lefty1基因真核表达载体构建鉴定及转染

Constuction and Identification of Lefty1 Gene Eukaryotic Expression Vector

目的:构建lefty1基因与pcDNA3.1(+)相融合的真核表达载体,并在lefty1基因的下游加上Flag标签,转染人肾小管上皮细胞株验证重组质粒活性。方法:设计并合成lefty1基因上下游引物,同时加上Flag标签序列,PCR扩增基因,并将其连接到真核表达载体pcDNA3.1(+)上,双酶切图谱分析和扩增产物测序鉴定所构建的真核表达载体。脂质体转染法将重组质粒pcDNA3.1(+)-lefty1-Flag和pcDNA3.1(+)分别转染至人肾小管上皮细胞株HK-2,qPCR和Western blot检测lefty1在mRNA和蛋白水平的表达。结果:以lefty1基因质粒模板扩增得到的片段约1 150bp,双酶切得到目的基因片段,测序的结果显示与lefty1基因序列相同。转染肾小管上皮细胞后,pcDNA3.1(+)-lefty1-Flag能表达lefty1蛋白。结论:成功构建lefty1基因真核表达载体,为一步研究lefty1基因功能奠定了基础。

Objective：To construct eukaryotic expression vector pcDNA3.1（＋）with lefty1 and add a Flag tag to the gene.Methods：lefty1was amplified by PCR,and identified by electrophoresis analysis.The PCR products and the eukaryotic expression vectors pcDNA3.1（＋）were digested by restriction endonuclesaes,then the digested lefty1 gene was inserted into the digested eukaryotic expression vector.After screening,the positive recombinants were picked out.PCR analysis,HindⅢand EcoRⅠrestriction endonucleases digest analysis and sequence analysis were used to identify the recombinants.pcDNA3.1（＋）-lefty1-Flag and pcDNA3.1（＋）were individually transfected into HK-2with lipofectamine.The expression of lefty1 was detected by qPCR and Western blot.Results：The 1 150 bp DNA fragment was amplified by PCR.The procucts of double restriction endonucleases degest analysis were 1 150 bp and 5.4kb DNA fragments.Sequence analysis showed that the lefty1 gene was inserted into pcDNA3.1（＋）vector.Lefty1 could be detected at mRNA and protein levels after the HK-2was transfected with pcDNA3.1（＋）-lefty1-Flag.Conclusion：The eukaryotic expression vector pcDNA3.1（＋）-lefty1 has been successfully constructed.