NLS-RARα蛋白在重组腺病毒Ad-NE感染的NB4中定位的验证

Location Verifi cation of NLS-RARα Protein in Infected NB4 Cell Line with Adenovirus Ad-NE

该实验主要验证重组腺病毒Ad．NE感染NB4细胞后，NLS-RARa蛋白的表达及其定位。用重组腺病毒Ad-NE感染NB4细胞，检测感染效率，分别用RT-PCR和Westernblot法在mRNA水平和蛋白水平验证转染成功：提取转染成功的NB4细胞的核蛋白，Westernblot法检测细胞核中NLS—RARα蛋白的表达；FITC—AnnexinV／DAPI双染色免疫荧光法检测转染成功的NB4细胞NLS-RARα的表达及定位；FITC—AnnexinV／PI双染色激光共聚焦法检测转染成功的NB4细胞中PNLS-RARα的表达及定位。结果显示，重组腺病毒Ad—NE和阴性对照腺病毒Ad-KZ对NB4细胞的感染效率可达70％～80％。RT-PCR和Westernblot结果显示，感染了重组腺病毒Ad—NLS-RAR的NB4细胞成功表龇基因和NE蛋白，且有NLS．RARa的蛋白表达。用细胞免疫荧光法、激光共聚焦法检测出已感染的NB4细胞中NLS—RARer蛋白的表达，并推测其主要定位于胞核。综上所述，该文成功用重组腺病毒Ad-NE感染NB4细胞，并用Westernblot法、免疫荧光法、激光共聚焦法验证了NLS-RARα蛋白的存在并推测其定位，为进一步研究急性早幼粒细胞白血病的早期诊断及复发监测提供了新的思路。

The experiment mainly verified the presence and location of NLS-RARα protein in NB4 cells infected with recombinant adenovirus Ad-NE. In our study, recombinant adenovirus Ad-NE was used to infect NB4 cells and then using FACS to confirm the efficiency of infection; RT-PCR and Western blot were used to identify the infection success in mRNA and protein levels; We extracted the nucleoprotein from successfully infected NB4 cells, and determined the expression of NLS-RARAα in NB4 cell nucleus by Western blot; FITC-Annexin V/DAPI double staining immunofluorescence and confocal laser scanning method were used to examine NLS-RARAα protein expression and localization in infected NB4 cells. The result showed that the infection efficiency of recom-binant adenovirus Ad-NE and negative control adenovirus Ad-KZ in NB4 cells were up to 70%-80%. The results of RT-PCR and Western blot showed that NE gene and protein were successfully expressed in NB4 cells infected with recombinant adenovirus Ad-NE, and there did exist NLS-RARa expression. Using cell immunofluorescence and confocal laser scanning, we found that NLS-RARα protein was present in infected NB4 cells and mostly local- ized in cell nucleus. In summary, we have successfully infected NB4 cells with recombinant adenovirns Ad-NE and verified the existence of NLS-RARα protein with Western blot, immunofluorescence and confocal laser scanning methods. NLS-RARα was mainly localized in nucleus. These results provided a new way for further study in early diagnosis and monitoring of acute promyelocytic myeloid leukemia.