[Construction and application of the replicon DNA recombinant plasmid pSVK-luc]

To study the expression of the replicon DNA vaccine in vivo by constructing a recombinant plasmid containing luciferase reporter gene on the basis of the Semiliki forest virus (SFV) replicon vector (pSVK-luc).

The luciferase gene fragment was amplified by PCR and cloned into the SFV replicon vector pSVK. The recombinant plasmid pSVK-luc was identified and screened by enzyme digestion and sequencing for the positive one. By chemical-based transfection, the positive plasmid was transferred into human embryonic kidney 293T cells to observe the expression of luciferase gene by flow cytometry and immunofluorescence. By electroporation, the plasmid was delivered into BALB/c mouse quadriceps femoris muscles to detect the expression of target gene by in vivo imaging system.

Sequencing revealed that the recombinant plasmid pSVK-luc we obtained was identical with the expected one. Flow cytometry and immunofluorescence showed that the expression of the luciferase gene in 293T cells and in vivo imaging system presented that the expression in the immunized mouse muscles.

A novel replicative DNA pSVK-luc has been successfully constructed, and can be expressed in 293T cells and in the muscle tissues of immunized mice, which provides a basis for future studies on the mechanism underlying the replicon DNA vaccine works in vivo and on the optimal electroporation conditions for the replicon DNA vaccine delivery in vivo.