ABSTRACT
Objective To detect the impact of reactive oxygen species ( ROS) on mitochondrial morphology and distri-bution during mitosis.Methods A viral vector in which the fluorescence gene was specifically under the control of mito-chondrial promoter was constructed and confirmed through DNA sequencing and Western blotting.After transfecting HeLa s3 cell with packaged virus, the HeLa s3-COX4tp-EGFP cell line stably expressing the mitochondrial fluorescence signal was obtained.With immunofluorescent staining, the impact of ROS on the morphology and distribution of mitochondria dur-ing mitosis was inspected.Result The cell line constantly expressing mitochondrial fluorescence signals was successfully constructed.Meanwhile,it was found that H2 O2 treatment could significantly change the morphology and distribution of mi-tochondria during mitosis by confocal microscopy.Conclusion Our study demonstrates that ROS can affect the morphology and distribution of mitochondria during mitosis.This research help study the relationship between the mitochondrial function and the regulation of mitosis in the future.
ABSTRACT
Objective To construct a prokaryotic expression vector pET-22b+with Middle East respiratory syndrome ( MERS) coronavirus nuclocapsid protein( NP) gene and to express and purify N protein.Methods N gene amplified by PCR was inserted into the prokaryotic expression vector pET-22b+.Recombinant plasmid was confirmed using DNA elec-trophoresis and sequencing.NP was expressed in E.coli BL21(DE3) by IPTG induced and purified by cation exchange chromatography using the AKTA purification system.Results The NP gene sequence was proved to be correct by sequen-cing and the protein was expressed in both soluble and insoluble forms in E.coli BL21 ( DE3 ) after IPTG induction.The purity and concentration of recombinant protein was improved obviously by cation exchange chromatography and enrich-ment.Conclusion Recombiant NP of high purity and concentration is purified and will facilitate NP functional research.