Aim: To obtain the intact encoding gene of human DC-SIGN and express its extracellular region in E.coli.
Methods: The intact cDNA encoding human DC-SIGN was amplified from total RNA of placenta of healthy parturient by RT-PCR, and its extracellular region was inserted into prokaryotic expression vector pET-41a. The recombinant plasmid pET-41a-sDC-SIGN was transformed into E.coli BL21(DE3). The expressed product was purified by GST affinity chromatography and identified by SDS-PAGE and Western blot.
Results: The DNA fragment of about 1 300 bp was amplified by RT-PCR, and cloned into pGM-T vector to obtain the recombinant plasmid pGM-DC-SIGN. The DNA fragment encoding the extracellular region of human DC-SIGN was amplified from pGM-DC-SIGN plasmid and the recombinant expression vector pET-41a-sDC-SIGN was constructed. The component of M(r); 66 000 in the purified recombinant product was found to be recognized by anti-DC-SIGN antibody.
Conclusion: The cDNA of human DC-SIGN is cloned and the protein of its extracellular region is obtained successfully, which lay the foundation for further research on functions of DC-SIGN.