Construction and expression of human DOC-1R retroviral expression vector in HeLa cells

Objective To construct retroviral expression vector carrying human DOC-1R gene and explore the recombinant protein expression in HeLa cells. Methods Eukaryotic expression vector pFLAG-DOC-1R was used as template, FLAG tagged-DOC-1R encoding fragment was amplified by PCR and subcloned into retroviral vector pLXSN. The identified recombinant DOC-1R vector was transfected into packaging cells GP-293 by liposome-mediated transfection to generate the retrovirus. The DOC-1R retrovirus was used to infect HeLa cells and overexpression of recombinant DOC-1R protein was demonstrated by SDS-PAGE analysis, Western blot and immunofluorescent stain. Results The clony PCR, double enzyme-digestion and DNA sequencing analysis demonstrated that recombinant retroviral expression vector pLXSN-FLAG-DOC-1R was constructed successfully. In HeLa cells, recombinant DOC-1R protein was detected with higher efficiency by retrovirus-mediated infection compared with that mediated by transfection with eukaryotic expression vector. Conclusion DOC-1R retroviral expression vector was constructed and the recombinant DOC-1R protein could be expressed efficiently in HeLa cells using the DOC-1R retroviral vector.