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人DC-SIGN全长编码区基因的克隆及其胞外段的原核表达 被引量：4

Cloning of the intact encoding gene of human DC-SIGN and prokaryotic expression of its extracellular region
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摘要 目的:克隆人DC-SIGN全长编码区基因,获得其胞外段的原核表达产物。方法:采用RT-PCR方法,从健康产妇胎盘中克隆DC-SIGN全长cDNA,扩增其胞外段基因并构建pET41a-sDC-SIGN重组表达质粒,在大肠杆菌BL21(DE3)中表达,以SDS-PAGE和Western blot鉴定表达产物。结果:从健康产妇胎盘总RNA中,扩增获得约1300bp的DNA片段,克隆至pGM-T载体获得重组质粒pGM-DC-SIGN。从pGM-DC-SIGN扩增DC-SIGN的胞外段基因,构建重组表达质粒pET-41a-sDC-SIGN;纯化表达产物sDC-SIGN-GST,鉴定其相对分子质量(Mr)为66000,Western blot证明其可与抗DC-SIGN抗体特异性结合。结论:成功克隆DC-SIGN全长编码区基因,并在大肠杆菌中成功表达其胞外段融合蛋白sDC-SIGN-GST,为进一步研究DC-SIGN的功能奠定了基础。 AIM： To obtain the intact encoding gene of human DC-SIGN and express its extracellular region in E. coIL 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 SDSPAGE 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 Mr 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.
作者 刘莹 左大明 卢晓 张丽芸 陈政良
机构地区 南方医科大学基础医学院免疫学教研室
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2009年第5期396-398,405,共4页 Chinese Journal of Cellular and Molecular Immunology
关键词 DC-SIGN 基因克隆 胞外区 蛋白表达 DC-SIGN gene cloning extracellular domain protein expression
分类号 R392.1 [医药卫生—免疫学]
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参考文献9

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二级参考文献26

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同被引文献27

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细胞与分子免疫学杂志
2009年 第5期

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