Expression, purification and biological activity analyses of soluble DC-SIGN
Objective To express and purify a recombinant DC -SIGN fusion protein in E.coli and analyze its biological activity. Methods DC-SIGN cDNA was amplified from mRNA of dendritic cells by RT-PCR and subsequently cloned into pcDNA3.1. Using pcDNA3.1-DC-SIGN as t emplate, the DC-SIGN lectin domain incorporated with KpnⅠ and SacⅠ was amplif ied by PCR and subcloned into prokaryotic expression vector pET-32a(+). Followi ng transfomation into E.coli AD494(DE3), expression was induced with IPTG. T he recombinant DC-SIGN fusion protein was purified with Ni 2+ -NTA agarose and identified by Western blot. Its biological activities were analyzed by HIV binding assay. Results Endonucleases digestion confirmed that lectin domain cDNA was inserted into the expression vector pET-32a(+). The recombinant expression vector successfully expressed DC-SIGN fusion protein in E.coli AD494(DE3). After purification by Ni 2+ -NTA agarose, the purit y of the DC-SIGN fusion protein was over 90%. Western blot indicated that it ha d a specific immunological activity with mouse anti human DC-SIGN antibody. Conclusion DC-SIGN fusion protein was successfully expresse d in E.coli AD494(DE3) and was purified by Ni 2+ -NTA agarose. HIV bind ing studies showed that the soluble DC-SIGN fusion protein inhibited both R5 an d X4 HIV binding to DC-SIGN.