学术
黑鲷（Acanthopagrus schlegeli）CXC型趋化因子的体外表达及其生物学活性
张勇[2] 蔡中华[1] 高春萍[1] 邢克智[3]
[1]清华大学深圳研究生院,深圳518055 [2]大连水产学院海水养殖与生物技术重点实验室,大连116023 [3]天津农学院养殖与生态重点实验室,天津300384
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pH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。[著者文摘]
文章出处:
《高技术通讯》-2008年18卷3期 -306-311页
栏目信息:
Recombinant expression and functional characterization of a CXC chemokine in black seabream （Acanthopagrus schlegeli ）
Zhang Yong, Cai Zhonghua, Gao chunping, Xing Kezhi （Graduate School at Shenzhen, Qinghua University, Shenzhen 518055） （ Key Lab of Mariculture and Biotechnology, Dalian Fishery University, Dalian 116023） （ Key Lab of Aqua-ecology and Aquaculture, Tianjin Agricultural University, Tianjin 300384）
Abstract:
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.[著者文摘]
Key words:
black bream （Acanthopagrus schlegeli）, ELR ^＋ CXC chemokines, recombination expression, biological activity
基金资助:
国家自然科学基金（30571432,40476057）、863计划（2006AA100311）、973计划（2006CB101805）和天津市科技创新能力与环境建设平台（06TXTJJC14200,05YFGPNC01900）资助项目.
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