Construction of the recombinant plasmid of human LMO1 gene and the expression and localization of fusion protein.

Objective To construct an eukaryotic expression vector of RNF6(ring finger protein 6)gene and identify its recombinant protein expression and localization.Methods Total mRNA was extracted from HEK-293 cells,cDNA was formed by reverse transcripton.The hRNF6 coding sequence was amplified by polymerase chain reaction(PCR) method and cloned into pcDNA3.1-myc-his A vector and pEGFP-C1 vector.After the target region was sequenced,the plasmid was transfected into HEK-293 cell lines.The expression of the recombinant plasmid in HEK-293 cells was proved by Western blot.The localization of pEGFP-RNF6 in CV-1 cell and gastric cancer cell SGC-7901 was observed by using laser scanning confocal microscopy.Results hRNF6 had been constructed into expressing vector pCDNA3.1-myc-his A and pEGFP-C1 successfully.The length of the fragment was 2 058 bp,identified by restriction enzymes digestion.The expression of myc-RNF6 fusion protein was detected by Western blot,with a molecular weight 78 kDa.The pEGFP-RNF6 protein was localized more in the nucleus,less in the cytoplasm.Conclusion The recombinant plasmid was successfully cloned into eukaryotic expressing vector,the pEGFP-RNF6 fusion protein was expressed mainly in the nucleus.