摘要
采用 DNA 体外重组技术,将一个黑鲷 CXC 趋化因子基因亚克隆到 pQE30表达载体并转化到大肠杆菌(M15)中诱导表达后,利用 pn 递降的缓冲液从金属亲和柱洗脱含6xHis 标签蛋白的方法得以纯化,纯化产物的分子量约为8kDa,表达量约占蛋白总量的25%,以6xHis 单抗为一抗,采用 Western-blotting 方法鉴定获得的蛋白为目的蛋白。对表达产物进行生物活性鉴定表明,该蛋白具有显著的嗜中性粒细胞和巨噬细胞的趋化活性(P<0.01,P<0.05),它对嗜中性粒细胞和头肾巨噬细胞的趋化能力呈剂量依赖性,且对嗜中性粒细胞的趋化能力大于对头肾巨噬细胞的作用,还能诱导改变嗜中性粒细胞和巨噬细胞对细菌的吞噬活性,但对嗜中性粒细胞吞噬能力的改变小于巨噬细胞。该趋化性因子在结构和功能上属不完全 ELR+CXC 型趋化因子,兼具 ELR^+CXC 和 ELR^-CXC 趋化因子的活性,是一种不成熟 ELR+CXC 型趋化因子。
With the method of DNA recombination in vitro, a black sea bream CXC （BSCXC） chemokine mature peptide coding sequence was sub cloned into expression vector pQE30 and expressed in E. Coli M15. About 8kDa expressed protein was purified by Ni-NTA chromatographic technology and identified by Western blotting using 6xHis tag antibodies. The bioactivity assay showed that the expressed protein had significantly chemotactic activities towards both leucocytes and macrophages in a dose dependent manner （ P 〈 0.01, P 〈 0.05）, and the BSCXC had stronger chemotaxis effects to leucocytes than that to macrophages. Furthermore, the BSCXC can significantly enhance the phagocytic activity of macrophages. The results indicate that the BSCXC is an incomplete ELR^＋ CXC chemokine for it is structurally and functionally incomplete, and possesses ELR^＋ CXC and ELR- CXC chemokine activities.
出处
《高技术通讯》
CAS
CSCD
北大核心
2008年第3期306-311,共6页
Chinese High Technology Letters
基金
国家自然科学基金(30571432,40476057)
863计划(2006AA100311)
973计划(2006CB101805)
天津市科技创新能力与环境建设平台(06TXTJJC14200,05YFGPNC01900)资助项目
关键词
黑鲷
ELR+CXC趋化因子
重组表达
生物活性
免疫应答
炎症反应
black bream （Acanthopagrus schlegeli）, ELR ^＋ CXC chemokines, recombination expression, biological activity
