[Construction and expression of a recombinant adenovirus with hEPO].

AIM

To construct the adenovirus vector containing human erythropoietin gene (hEPO) and to detect its expression in HeLa cells.

METHODS

hEPO gene was subcloned into the shuttle plasmid pAdTrack-CMV and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with hEPO. The recombinant adenoviral plasmid with hEPO was digested with Pac I and then transfected into HEK293 cells to package recombinant adenovirus particles. The recombinant adenovirus containing hEPO gene was identified by PCR and transmission electron microscopy and was purified by cesium choride density centrifugation. The viral titer was checked by GFP. HeLa cells were infected by the recombinant adenovirus and the transcription and expression of hEPO gene were analyzed by RT-PCR and Western blot.

RESULTS

Recombinant adenovirus vector pAdEasy-hEPO was constructed and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAdEasy-hEPO. The high expression of green fluorescence protein expression in HEK293 and HeLa cell lines was observed under fluorescent microscope. PCR and electron microscopy test showed that a recombinant adenovirus RAd-hEPO was successfully constructed and the titer of the recombinant adenovirus reached 1.8 x 10(10) pfu/mL.The expression of hEPO gene in the infected HeLa cells was confirmed by Western blot.

CONCLUSION

The recombinant adenovirus RAd-hEPO can be successfully expressed in the infected HeLa cells, which lays the foundation for further research into therapy for ischemic heart diseases.