Construction of eukaryotic expression vector pEGFP-N1-MKRN1 and its expression in Siha cells
ABM: To construct eukaryotic expression vector of pEGFP-Nl-MKRNl and obtain positive Siha cell clones expressing MKRN1 stably, and to establish a cell model for studying the effect of MKRN1 on cell proliferation and apoptosis. METHODS:The cDNA of human MKRN1 was amplified by RT-PCR from the total RNA isolated from the 293 cells and was inserted into pEGFP-Nl vector to construct the recombinant eukaryotic expression vector pEGFP-N1-MKRN1. The recombinant plasmid was thentransferred into Siha cells by liposome. Overexpression of the MKRN1 in the transfected Siha cells was confirmed with Western blot. RESULTS: By the use of RT-PCR, a 1477 bp DNA fragment was successfully amplified from the 293 cells, which matched the length of cDNA encoding the MKRN1. Enzyme digestion analysis and DNA sequencing showed that the target gene was cloned into recombinant vector. Green fluorescence was emitted from transfected cells under fluorescent microscope. Western blot analysis revealed the fusion protein pEGFP-N1-MKRNl could be expressed stably in the transfected Siha cells. CONCLUSION: The eukaryotic expression pEGFP-N1-MKRNl was successfully constructed. The positive Siha cell clones expressing MKRN1 stably were obtained, which have provided solid foundation and a cell model for further research on the function of MKRNl and its relationship with cell proliferation and apoptosis.
MKRN1pEGFP-Nl vectorSiha cellproliferation and apoptosis
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维普
