Construction of DC-SIGN eukaryotic expression vector and establishment of BHK21 cell line stably transfected by the recombinant vector / 解放军医学杂志

ABSTRACT

Objective To construct a eukaryotic expressing vector harboring human DC-SIGN, and establish a BHK21 cell line stably and highly expressing DC-SIGN. Methods The DC-SIGN gene fragment which contained Not I and BamH I sites was amplified by PCR from pUNO-hDCSIGN1Aa plasmid, digested with Not I and BamH I, and then cloned into an eukaryotic expression vector pIRES-neo to construct eukaryotic expression vector pIRES-neo-DC-SIGN. The recombined plasmid was identified with Not I and BamH I enzyme digestion and sequencing, the latter was then transfected to BHK21 cells by LipofectamineTM 2000. After screening culture by G418, BHK21 cell line stably expressing DC-SIGN was established. The expression of DC-SIGN was identified by flow cytometry, Western blotting and immunofluorescence method. Results The gene sequence of DC-SIGN was consistent with that of design. PCR and double enzyme digestion analysis showed that the recombinant plasmid pIRES-neo-DC-SIGN was constructed successfully. After transfection, positive clones were selected with G418. After limiting dilution assay, BKH21 cell lines stably expressing DC-SIGN were established. The detection result of flow cytometry showed that the expression ratio of DC-SIGN positive clones was close to 90%. The result of immunofluorescence displayed that the expression of DC-SIGN was mostly located on the surface of cell membrane. Western blotting displayed the specific band of DC-SIGN protein. It showed that the BHK21 cells stably expressing DC-SIGN were successfully established. Conclusion DC-SIGN eukaryotic expression vector has been successfully constructed. The successful establishment of BHK21 cell lines which can stably express DC-SIGN provides a substantial foundation for further study on the DC targeting vaccines.