携带突变型人IκBαDN cDNA重组嵌合腺病毒Ad5F35的制备与表达

Generation and expression of recombinant chimeric Ad5F35 adenovirus carrying mutant human IκBαDN cDNA

目的制备携带突变型人核转录因子κB抑制蛋白α(IκBα)cDNA的重组5型和35型嵌合腺病毒(Ad5F35),并分析其表达。方法应用DNA重组技术,从含有突变型人IκBα(IκBαDN)cDNA的重组逆转录病毒载体pCLX-IκBαDN克隆IκBαDNcDNA到pDC316载体以构建pDC-IκBαDN并随即制备携带IκBαDNcDNA的重组嵌合腺病毒Ad5F35。转染HL-60细胞后,通过实时定量PCR分析IκBα的表达。结果通过DNA重组技术,将IκBαDNcDNA成功克隆到pDC316载体。DNA序列测定显示,重组pDC-IκBαDN载体内携带有无其它突变并具有正确阅读框架的人IκBαDNcDNA。转染HL-60细胞48h后,HL-60细胞IκBαmRNA表达增加。结论成功制备到含有人IκBαDNcDNA的重组嵌合腺病毒Ad5F35,并能够在HL-60细胞有效表达IκBα。

Objective To generate recombinant adenovirus fused with type 5 and 35 adenovirus （Ad5F35）,in which mutant human inhibitor-α of nuclear transcription factor κB （IκBα） cDNA was carried. Methods pDC-IκBαDN vector was constructed by cloning mutant human IκBα（IκBαDN） cDNA from the recombinant retroviral vector of pCLX-IκBαDN that contains the IκBαDN cDNA by DNA recombination technique. Subsequently, the recombinant chimeric Ad5F35 adenovirus carrying the IκBαDN cDNA was produced. After transferring HL-60 cells, IκBα mRNA expression was analyzed by real time PCR. Results IκBαDN cDNA was successfully cloned into the vector of pDC316 by DNA recombination technique. DNA sequencing result indicated that the recombinant vector of pDC-IκBαDN carried IκBαDN cDNA that contained correct reading-frame of IκBαDN cDNA, and no any other mutation. The expression of IκBα mRNA was increased in HL-60 cells 48 hours after transduction. Conclusion The recombinant chimeric Ad5F35 adenovirus carrying human IκBαDN cDNA was generated,and the IκBα mRNA was effectively expressed in HL-60 cells.