Construction of eukaryotic expression vector pEGFP-VASP-lC and its expression in HeLa cells

Objective: To construct eukaryotic expression vector pEGFP-VASP-ZC and obtain positive HeLa cell clones expressing lC region of human VASP stably, and to establish a cell model for exploring the role of lC region-mediated oligomerization of VASP in the the migration of HeLa cells further. Methods: The cDNA encoding the lC region of human VASP was amplified by RT-PCR from the total RNA isolated from the human ECV304 cells and was inserted into pEGFP-C1 vector to construct the recombinant eukaryotic expression vector pEGFP-VASP-ZC. The recombinant plasmid was then transferred into HeLa cells by liposome. Overexpression of the ZC region of human VASP in the transfected HeLa cells was confirmed with Western blot. Results: By the use of RT-PCR, a 310 bp DNA fragment was successfully amplified from the human ECV304 cells, which matched the length of cDNA encoding the ZC region of human VASP. 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 EGFP-VASP-lc could be expressed stably in the transfected HeLa cells. Conclusion: The eukaryotic expression pEGFP-VASP-lC was successfully constructed. The positive HeLa cell clones expressing the lC region of human VASP stably were obtained, which may be a cell model for studying the role of lC region-mediated oligomerization of VASP in the migration of HeLa cell further.