可溶性DC-SIGN的表达、纯化及其生物学活性分析  

Expression, purification and biological activity analyses of soluble DC-SIGN

作　　者：马新锋[1] 胡勤学[1] 崔天盆[2] 杨继红[3] 张治平[1] 

机构地区：[1]中国科学院武汉病毒研究所,430071 [2]武汉协和医院 [3]武汉市疾病预防控制中心

出　　处：《中华微生物学和免疫学杂志》2004年第1期67-71,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目 (项目编号 30 0 80 0 0 830 2 70 6 46 )

摘　　要：目的　在大肠杆菌中表达并纯化DC SIGN融合蛋白并对该融合蛋白的抗原特异性和生物学活性进行分析。方法　以重组质粒pcDNA3 .1 DC SIGN为模板 ,进行PCR扩增出带KpnⅠ和SacⅠ酶切位点的人DC SIGN凝集素cDNA ,经相应酶切后插入原核表达载体pET 3 2a(+ ) ,转化大肠杆菌AD494(DE3 ) ,经IPTG诱导表达DC SIGN融合蛋白 ,用Ni2 + NTA树脂对融合蛋白进行纯化 ,以Westernblot试验进行鉴定 ,通过HIV与DC SIGN的亲和实验研究融合蛋白的生物学活性。结果　酶切鉴定证实DC SIGN凝集素基因已插入原核表达载体pET 3 2a(+ )。重组表达质粒pET 3 2a(+ ) DL在大肠杆菌AD494(DE3 )中成功表达了DC SIGN融合蛋白 ,其相对分子质量 (Mr)约为 3 5× 10 3。Ni2 + NTA树脂纯化后 ,融合蛋白的纯度可达 90 %以上。Westernblot试验显示DC SIGN融合蛋白与鼠抗人DC SIGN抗体有特异性免疫反应。结论　在大肠杆菌中表达DC SIGN融合蛋白 ,用亲和层析的方法对其进行初步纯化 ;HIV与DC SIGN的亲和实验表明 ,可溶性DC SIGN融合蛋白能抑制R5和X4HIV与DCObjective To express and purify a recombinant DC -SIGN fusion protein in E.coli and analyze its biological activity. Methods DC-SIGN cDNA was amplified from mRNA of dendritic cells by RT-PCR and subsequently cloned into pcDNA3.1. Using pcDNA3.1-DC-SIGN as t emplate, the DC-SIGN lectin domain incorporated with KpnⅠ and SacⅠ was amplif ied by PCR and subcloned into prokaryotic expression vector pET-32a(+). Followi ng transfomation into E.coli AD494(DE3), expression was induced with IPTG. T he recombinant DC-SIGN fusion protein was purified with Ni 2+ -NTA agarose and identified by Western blot. Its biological activities were analyzed by HIV binding assay. Results Endonucleases digestion confirmed that lectin domain cDNA was inserted into the expression vector pET-32a(+). The recombinant expression vector successfully expressed DC-SIGN fusion protein in E.coli AD494(DE3). After purification by Ni 2+ -NTA agarose, the purit y of the DC-SIGN fusion protein was over 90%. Western blot indicated that it ha d a specific immunological activity with mouse anti human DC-SIGN antibody. Conclusion DC-SIGN fusion protein was successfully expresse d in E.coli AD494(DE3) and was purified by Ni 2+ -NTA agarose. HIV bind ing studies showed that the soluble DC-SIGN fusion protein inhibited both R5 an d X4 HIV binding to DC-SIGN.

关 键 词：DC-SIGN 原核表达 生物学活性 大肠杆菌 融合蛋白 

分 类 号：R346[医药卫生—基础医学]