论文部分内容阅读
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。
OBJECTIVE: To clone the full length coding region of human DC-SIGN and obtain the prokaryotic expression product of its extracellular domain. Methods: The full-length cDNA of DC-SIGN was cloned from healthy maternal placenta by RT-PCR. The extracellular domain of the DC-SIGN gene was amplified and the recombinant plasmid pET41a-sDC-SIGN was constructed and expressed in E. coli BL21 SDS-PAGE and Western blot to identify the expression product. Results: From the healthy maternal placental total RNA, a DNA fragment of about 1300bp was amplified and cloned into the pGM-T vector to obtain the recombinant plasmid pGM-DC-SIGN. The recombinant plasmid pET-41a-sDC-SIGN was constructed by amplifying the extracellular domain of DC-SIGN from pGM-DC-SIGN. The expressed product sDC-SIGN-GST was purified and its molecular weight was identified as 66,000 Western blot confirmed that it can specifically bind to anti-DC-SIGN antibody. CONCLUSION: The full-length coding region of DC-SIGN was cloned successfully and its extracellular fusion protein sDC-SIGN-GST was successfully expressed in E. coli, which laid the foundation for further study on the function of DC-SIGN.