骨桥蛋白基因质粒的构建及慢病毒包装的实验研究

The construction of osteopontin gene recombinant plasmid and its package of lentivirus

摘要: 目的:构建骨桥蛋白(OPN)基因的重组质粒,并包装慢病毒。方法:从C57BL/6野生型小鼠肾脏组织提取总RNA,逆转录为cDNA经特定引物扩增出OPN基因片段,并大量扩增,利用基因重组技术该基因片段与慢病毒载体pCDH-CMV-MCS-EF1-GFP-T2A-Puro连接形成重组质粒,该重组质粒转染293T细胞进行慢病毒包装后测定滴度。结果:DNA测序证实提取的基因为OPN片段,慢病毒滴度达2.5×10⁸ TU/ml。结论:成功包装了含有OPN基因片段的慢病毒,为进一步研究OPN对各种细胞的生物学功能的影响奠定了基础。

Abstract: Objective: To construct the recombinant plasmid containing the osteopontin(OPN) gene and package lentivirus with it.Methods: The total RNA of kidney tissue was abstracted from C57BL/6 wild type mouse,and then was transcribed to cDNA.The OPN gene was largely amplified with special primers,and then cloned to lentivirus carrier to constract the pCDH-CMV-MCS-EF1-GFP-T2A-Puro.The lentivirus was packaged by transfecting the plasmids into 293T cells,and the virus titer was tested.Results: The OPN gene fragment was successfully cloned by sequencing.The virus titer was 2.5×10⁸ TU/ml.Conclusions: The lentivirus containing OPN gene is successfully packaged,which lays the foundation in further researching the biological function of OPN gene.