CD74-ROS1-L1951R点突变质粒的构建与鉴定

Construction and verification of CD74-ROS1-L1951R point-mutated plasmid

目的通过重叠延伸PCR技术构建CD74-ROS1-L1951R点突变质粒。方法根据CD74-ROS1融合基因序列和真核表达质粒p CM V-C-Flag上的多克隆位点设计引物,利用重叠延伸PCR技术对CD74-ROS1融合基因进行基因定点突变得到CD74-ROS1-L1951R点突变融合基因,随后将其酶切定向连入真核表达质粒p CMV-C-Flag启动子下游,构建p CMV-C-Flag-CD74-ROS1-L1951R重组质粒。重组质粒经菌落PCR、双酶切鉴定和测序,鉴定目的基因是否插入p CM V-CFlag质粒及定点突变是否成功。结果通过双酶切鉴定和测序结果显示,质粒的方向及序列正确,阅读框正确无误,表明CD74-ROS1-L1951R基因成功插入p CMV-C-Flag质粒,且CD74-ROS1-L1951R基因定点突变正确。结论成功构建了p CM V-C-Flag-CD74-ROS1-L1951R真核表达重组质粒,为研究CD74-ROS1点突变导致的耐药机制奠定了基础,可用于后续研究。

Objective To construct the recombinant plasmid carrying CD74-ROS1-L1951 R fusion gene.Methods Design primers based on the CD74-ROS1 fusion gene sequence and the multiple cloning site on the eukaryotic expression plasmid p CMV-C-Flag.The L1951 R point mutation was site-directed mutation which was constructed by overlapping extension PCR.Then CD74-ROS1 fusion gene was inserted into the carrier promoter downstream of vector p CMV-C-Flag to build the p CMV-C-Flag-CD74-ROS1-L1951 R recombinant plasmid.The recombinant plasmid was verified by restriction enzyme digestion and gene sequencing.Results The inserted part of pCMV-C-Flag-CD74-ROS1-L1951 R plasmid showed that the direction and sequence pCMV-C-Flag-CD74-ROS1-L1951 R plasmid and the reading frame were correct.The results proved that the CD74-ROS1-L1951 R point mutation was correct.Conclusion pCMV-C-Flag-CD74-ROS1-L1951 R eukaryotic expression vector was successfully constructed,which can be used for the following-up study.Our experiment laid a foundation for further mechanism study on CD74-ROS1 mutation-leaded resistance.