人转铁蛋白受体基因慢病毒表达载体的构建和鉴定

Construction and identification of h TfR lentivirus expression vector

目的将人转铁蛋白受体(hTfR)基因克隆到慢病毒表达载体pLENTI6.3,并鉴定其正确性,为活体干细胞MR分子成像提供实验基础。方法利用聚合酶链反应技术(PCR)扩增hTfR基因,并克隆到pLENTI6.3载体;经过菌落PCR初步筛选、Bam H1酶切鉴定和测序鉴定构建的pLENTI6.3-hTfR-IRES-EGFP重组载体。结果通过PCR技术成功扩增了hTfR基因;hTfR基因片段重组到pLENTI6.3载体;菌落PCR和酶切鉴定,凝胶电泳结果均显示得到和理论大小相符的片段;DNA测序结果与Genbank序列完全一致。结论 pLENTI6.3-hTfR-IRES-EGFP慢病毒表达载体构建成功,为下一步进行活体干细胞MR分子成像实验研究奠定基础。

Objective To clone human transferrin receptor （hTfR） into lentivirus expression vector-pLENTI6.3 and identify the reconstructed plasmid, which will provide experimental basis for in vivo stem cell magnetic resonance （MR） molecular imaging . Methods The hTfR gene cDNA was amplified by polymerase chain reaction （PCR） and cloned into pLENTI6.3 vector. The reconstructed pLENTI6.3-hTfR-IRES-EGFP plasmid was identified by colony PCR , Bamhl enzyme digestion and DAN sequence analysis. Results The hTfR gene fragment was constructed into pLENTI6. 3 vector. The reconstructed plasmid was identified by colony PCR, enzyme digestion and DAN sequence analysis, which showed that hTfR gene sequence was exactly the same as that in Genbank. Conclusion The pLENTI6.3-hTfR-IRES-EGFP lentivirus expression vector is successfully constructed, which provides experimental basis for experimental researches about in vivo stem cell MR molecular imaging.