DC-SIGN胞外区基因的克隆、蛋白表达及抗体制备  

Cloning,Expression and Antibody Preparation of Extracellular Region of DC-SIGN

作　　者：吕佰瑞[1] 刘朝奇[1] 

机构地区：[1]三峡大学分子生物学研究所,宜昌443002

出　　处：《生物技术通报》2011年第2期187-191,共5页Biotechnology Bulletin

摘　　要：旨在构建DC-SIGN胞外区基因原核表达质粒,诱导蛋白表达并制备多克隆抗体。用PCR的方法扩增编码DC-SIGN胞外区的基因序列,将其克隆到原核表达载体pET-28a(+)中,利用大肠杆菌表达系统表达DC-SIGN胞外区蛋白,用H is抗体做W estern Blotting鉴定目的蛋白的免疫原性。用纯化的DC-SIGN胞外区蛋白免疫日本大耳白兔,制备多克隆抗体。通过酶联免疫吸附试验(ELISA)检测抗体效价,免疫荧光法检测其特异性。结果显示,原核表达载体pET-28 a(+)-DC-SIGN胞外区基因成功构建,可在大肠杆菌BL21(DE3)中高效表达,获得相对分子质量约20 kD的DC-SIGN胞外区蛋白,经Westernb lotting鉴定为正确。纯化后的蛋白免疫大耳白兔,制备的多克隆抗体具有较强免疫原性和特异性。本研究得到了纯化的DC-SIGN胞外区蛋白,并制备了具有特异性和高效价的抗体,为研究DC-SIGN生物学功能提供试验基础。This study was to construct expression plasmid pET-28a（＋）/DC-SIGN extracellular domain and prepare its polyclonal antibody.The gene fragment of extracellular region of DC-SIGN was amplified by PCR and cloned into pET-28a（＋） prokaryotic expressing vector.E.coli BL21（DE3）transformed with pET-28a（＋）-DC-SIGN was induced by IPTG and the expressed protein was detected through Western Blotting.The purified protein was used to immunize rabbits to prepare polyclonal antibody.The titer and specificity of rabbit anti-serum were assayed by ELISA and immunofluorescence assay.Results showd that we constructed pET-28a（＋）-DC-SIGN successfully.DC-SIGN protein could be expressed in E.coli BL21（DE3）with high efficiency,and it was a kind of 20 kD molecular weight protein.We got correct responding antibody through immunizing rabbit with the purified protein.The polyclonal antibody could combine purified protein with a high titer by ELISA.Immunofluorescence assay showed that the antibody could specifically bind to DC-SIGN expressed in eukaryotic cells.DC-SIGN extracellular domain was cloned and expressed in prokaryotic cell.The purified protein and its antibody could provide the basis for studying biological functions of DC-SIGN.

关 键 词：DC-SIGN 蛋白表达 多克隆抗体 Western BLOTTING 免疫荧光 

分 类 号：Q78[生物学—分子生物学]