MOB2基因在真核细胞中的表达研究

通过RT-PCR从培养的HUVECs中扩增MOB2基因片段,将该片段克隆在真核表达载体pEGFP-C1中,转染NIH3T3细胞,经G418筛选,构建稳定表达细胞系,并用荧光显微镜和Western blot对其进行鉴定。经RT-PCR扩增出734 bp基因片段,经测序分析并与GenBank的DNA序列比对分析后,在NIH3T3细胞中表达。G418筛选后,细胞荧光信号较强,Western blot检测,细胞中的融合蛋白能够与抗MOB2的多抗结合。成功地扩增了HUVECs中的MOB2基因全长cDNA并进行真核表达与鉴定。

Eukaryotic expression of Mps one binder 2 (MOB2)

To clone and construct the eukaryotic expression and verification of MOB2 gene, MOB2 gene fragment was amplified by reverse transeription-polymerase chain reaction （RT-PCR） from human umbilical vein endothelial cells （HUVECs） and subcloned into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid was transfected into NIH3T3 cell, and the positive clones were screened by G418 to establish the stable expression cell line. Fluorescence microscope and Western blotting assays were used to confirm over-expression of MOB2 protein in the transfected cells. RT-PCR yielded a fragment of 734 bp, and the inserted MOB2 sequence was verified by sequence analysis and sequence alignment with DNA sequence in Genbank. The fusion protein was observed in NIH3T3 cells after G418 selection under the fluorescence microscope, and confirmed combination with polyclonal antibody against MOB2 by Western blotting assay. Full-length cDNA of MOB2 from t HUVECs was successfully amplified, and an eukaryotic expression vector of MOB2 was successfully established. This method could provide a good tool for us to further study the functional involvement of MOB2.