Deng Wen-Wen, Liu Shu-Kun, Zhang Zun-Zhen
Department of Environmental Health, West China School of Public Health, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2013 Mar;44(2):170-4.
To construct the eukaryotic express vector containing apoptosis-inducing factor (AIF) gene and to study its expression in A549 cells.
According to the GenBank AIF mRNA sequence, specific primers to amplify AIF gene from lung carcinoma cell line A549 by RT-PCR was designed. The amplified AIF gene fragment was cloned into plasmid pUC-T by TA cloning, then double enzyme digestion and DNA sequencing were used to identifying the positive recombinant AIF-pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1(+). The positive recombinant AIF-pcDNA3.1(+) was transfected into A549 cells, and expression of AIF gene was verified by RT-PCR and Western blot.
AIF target gene was successfully amplified and cloned into the pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1(+), and it was completely coincided with the AIF sequence in GenBank suggested by cells transfected with AIF-pcDNA3. 1(+) was much higher than that of control cells which was not transfected with AIF-pcDNA3.1(+).
The AIF eukaryotic expression vector AIF-pcDNA3.1(+) is successfully constructed in A549 cells and it could be experimental foundations for further study of AIF gene.
构建含凋亡诱导因子（AIF）基因的真核表达载体，并研究其在A549细胞中的表达。
根据GenBank中AIF mRNA序列，设计特异性引物，通过RT-PCR从肺癌细胞系A549中扩增AIF基因。将扩增的AIF基因片段通过TA克隆克隆到质粒pUC-T中，然后用双酶切和DNA测序鉴定阳性重组体AIF-pUC-T。回收目的片段并克隆到真核表达载体pcDNA3.1(+)中。将阳性重组体AIF-pcDNA3.1(+)转染到A549细胞中，通过RT-PCR和Western blot验证AIF基因的表达。
成功扩增AIF目的基因并克隆到pUC-T中。回收目的片段并克隆到真核表达载体pcDNA3.1(+)中，与GenBank中的AIF序列完全一致。转染AIF-pcDNA3.1(+)的细胞中AIF的表达明显高于未转染AIF-pcDNA3.1(+)的对照细胞。
成功构建了AIF真核表达载体AIF-pcDNA3.1(+)，为进一步研究AIF基因奠定了实验基础。
