人KLF4基因克隆及重组KLF4慢病毒表达载体的构建

Clone of human KLF4 gene and construction of recombinant KLF4 lentiviral vector

背景:KLF4基因在细胞分化过程中有重要作用。目的:构建KLF4基因慢病毒过表达载体。方法:聚合酶链反应扩增(PCR)目的基因KLF4后插入慢病毒表达载体pCDH中,构建重组载体pCDH-KLF4。通过PCR、双酶切和DNA测序方法对其鉴定,共同转染293NT细胞包装病毒,荧光显微镜观察报告基因的表达情况,收集病毒上清,测定病毒的滴度。将重组的慢病毒感染5637细胞,RT-PCR检测KLF4mRNA的表达。结果与结论:重组慢病毒表达载体质粒经限制性酶切和DNA测序分析证实重组慢病毒载体pCDH-KLF4的插入序列完全正确,重组慢病毒载体感染5637细胞后,细胞内KLF4mRNA高表达。结果证实,实验成功构建KLF4基因慢病毒过表达载体。

BACKGROUND： KLF4 is an essential gene for celt differentiation OBJECTIVE： To construct a lentiviral vector pCDH-KLF4 gene. METHODS： KLF4 gene amplification was used by PCR. Gene amplification products were inserted into the ientiviral vector pCDH and to co-transfect 293TN cells. DNA sequencing was used to confirm the recombinant vector. The virus supernatant was harvested and titrated. The expression of KLF4 was detected by reverse transcription-PCR. RESULTS AND CONCLUSION： DNA sequencing confirmed that the sequence of amplified KLF4 gene was consistent with the Genebank data. In transfected 5637 cells, KLF4 mRNA was over-expressed. Results verified that lentiviral vevtors of KLF4 gene over-expression were successfully constructed.