摘要
目的探讨Oct4,Sox2,c-Myc及Klf4-慢病毒载体的构建与包装。方法从含有Oct4,Sox2,c-Myc及Klf4的质粒中获取目的基因,将目的基因与酶切线性化的慢病毒载体进行定向连接,其连接产物转化细菌感受态细胞,对长出的阳性克隆进行测序和比对分析,并对长出的阳性克隆进行PCR鉴定,通过三质粒系统共转染293T细胞对慢病毒进行包装并进行滴度测定。结果成功构建并包装出Oct4,Sox2,c-Myc及Klf4-慢病毒载体,基因测序结果与目标序列完全一致;PCR鉴定结果显示,4个目的基因均为阳性;Oct4,Sox2,c-Myc及Klf4-慢病毒载体滴度分别为1×109,1×109,1×109,2×109 TU/mL。结论通过三质粒系统可构建并包装Oct4,Sox2,c-Myc及Klf4-慢病毒载体。
Objective To explore the construction and packing of lentivirus vectors including Oct4, Sox2, c-Myc and Klf4. Methods The objective genes were acquired from the plasmid consisting Oct4, Sox2, c-Myc and Klf4, and were directionally connected to the enzyme linearized lentivirus vectors. The production was transformed into competent bacteria. The positive clones were sequenced and compared when positive objective plasmid was successfully constructed with Lise PCR technique. Three-plasmid system was used to transfect 293T cells for packing lentivirus and testing the titer of lentivirus. Results Lentivirus vectors were successfully constructed and packed including Oct4, Sox2, c Mye and Klf4. The sequence of gene was consistent with objective sequence. Oct4, Sox2, c-Myc and Klf4 were expended positively with PCR technique. The titers of Oct4, Sox2, c-Myc and Klf4 were 1 ×10^9 , 1 ×10^9 , 1 ×10^9 and 2 ×10^9 TU/mL. Conclusion Three-plasmid system can successfully construct and pack lentivirus vectors including Oct4, Sox2, c-Myc and Klf4.
出处
《中华实用诊断与治疗杂志》
2012年第8期766-767,770,共3页
Journal of Chinese Practical Diagnosis and Therapy
关键词
慢病毒载体
构建
目的基因
包装
Lentivirus vector
construction
objective gene
packing
