人可溶性DC-SIGN的原核表达及鉴定

Prokaryotic expression and identification of human soluble DC-SIGN

目的构建可溶性DC-SIGN(sDC-SIGN)原核表达载体,获得不含标签蛋白的sDC-SIGN蛋白。方法采用PCR方法,从含人DC-SIGNcDNA的重组质粒pGM-DC-SIGN扩增DC-SIGN胞外区基因片段,插入原核表达载体pET17b,构建重组表达载体pET17b-sDC-SIGN,经酶切图谱和测序鉴定,转入E.coliBL21(DE3)诱导表达蛋白,用抗人DC-SIGN抗体-Sepharose4B亲和层系纯化表达产物,以SDS-PAGE和Westernblot鉴定。结果从重组质粒pGM-DC-SIGN扩增获得1300bp目的基因片段,构建重组表达质粒pET17b-sDC-SIGN,其酶切图谱和序列与预期相符。纯化表达产物sDC-SIGN,鉴定其分子质量为38000,Westernbolt证明其可与抗人DC-SIGN抗体特异性结合。结论获得了能高效表达重组人sDC-SIGN的大肠杆菌菌株和不带任何标签蛋白的sDC-SIGN蛋白,为深入研究sDC-SIGN的功能奠定了基础。

Objective To express the soluble form of human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin （sDC-SIGN） without any tag protein in E. coli. Methods The target sequence in pGM-DC-SIGN plasmid that contains human DC-SIGN cDNA was amplified by PCR, inserted into prokaryotic expression vector pET17b and identified by restriction mapping and sequencing. The recombinant expression vector was transformed into E. coli BL21 （DE3） cells. The expressed product was purified by anti-DC-SIGN mAb-Sepharose 4B Affinity Chromatography and identified by SDS-PAGE and Western blot assay. Results The DNA fragment of 1 300 bp, which encode the extracellular region of human DC-SIGN, was amplified from pGM-DC-SIGN plasmid and the recombinant expression vector pET-17b-sDC-SIGN was constructed and confirmed by restriction maps and sequencing. The component of Mr 38 000 in the purified recombinant product was detected by SDS-PAGE and could be recognized by anti-DC-SIGN antibodies in Western blot. Conclusion The recombinant human sDC-SIGN protein was expressed without any tag and purified. This work lays the foundation for further research on functions of sDC-SIGN.