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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 construct the prokaryotic expression vector of soluble DC-SIGN (sDC-SIGN) and obtain the sDC-SIGN protein without tagging protein. Methods The extracellular region of DC-SIGN gene was amplified by PCR from the recombinant plasmid pGM-DC-SIGN containing human DC-SIGN cDNA and inserted into the prokaryotic expression vector pET17b to construct the recombinant expression vector pET17b-sDC-SIGN. The expressed product was transformed into E.coli BL21 (DE3) and purified by anti-human DC-SIGN antibody-Sepharose 4B affinity chromatography. The expressed product was identified by SDS-PAGE and Western blot. Results The 1300bp gene fragment was amplified from the recombinant plasmid pGM-DC-SIGN, and the recombinant plasmid pET17b-sDC-SIGN was constructed. Its digestion pattern and sequence were consistent with the expected results. The expression product sDC-SIGN was purified and its molecular mass was identified as 38,000. Westernbolt proved that it could specifically bind to anti-human DC-SIGN antibody. Conclusion Escherichia coli strains with high expression of recombinant human sDC-SIGN and sDC-SIGN protein without any tagged protein were obtained, which laid the foundation for further study on the function of sDC-SIGN.