重组人可溶性CD14在昆虫细胞表达系统中的表达  

Expression of human soluble CD14 in baculovirus expression system

作　　者：张杰[1] 薛沿宁[1] 郑莉[1] 龙建银[1] 刘农乐[1] 王芳[1] 王会信[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850

出　　处：《生物技术通讯》2001年第3期184-187,共4页Letters in Biotechnology

摘　　要：BAC TO BAC 杆状病毒表达系统是一种快速、高效、便捷的表达系统。将人可溶性CD14(sCD14)基因克隆入pFASTBAC1转移质粒中 ,重组质粒转化DH10BAC 感受态细胞 ,目的基因通过同源重组插入BacmidDNA中 ,后者转染sf2 1昆虫细胞获得重组杆状病毒。利用重组蛋白C 末端的 6×His·Tag ,经TALON金属螯合色谱将重组病毒感染昆虫细胞获得的无血清培养上清一步纯化得到重组蛋白 ,计算表明从 1L培养基中可纯化到约 8mg纯度大于95 %的重组sCD14蛋白 ,免疫印迹结果表明重组蛋白具有与抗 6×His单抗和抗CD14单抗结合的抗原性。凝胶迁移实验和细胞活性实验表明重组sCD14蛋白能在体外与LPS结合 ,并能增强LPS诱导THPHuman soluble CD14 gene were inserted into pFASTBAC1 vector and the recombinant plasmids were transformed into DH10BAC competent cells for transposition into the bacmid. After the recombinant bacmids had been determined to be correct, the isolated bacmid DNAs were transfected into sf21 insect cells and recombinant baculovirus stocks were harvested from the transfected cells. The yield of purified recombinant proteins were 8mg per liter cultural supernatant after passing TALON metal affinity resin. Western Blot showed that the purified proteins could bind to both anti 6×His McAb and anti-human CD14 McAb and gel shift assay suggested that recombinant sCD14 could bind to LPS, and they could also increase cytotoxic factors production of LPS-induced THP-1 cells.

关 键 词：CD14 细菌脂多糖 杆状病毒 昆虫细胞表达系统 

分 类 号：R394.8[医药卫生—医学遗传学] R392.11[医药卫生—基础医学]