虹鳟lect2 的酵母表达、纯化及生物活性分析.pdf

动 物 学 研 究 2013，Feb. 34(1): 33−38 CN 53-1040/Q ISSN 0254-5853 Zoological Research DOI ：10.3724/SP.J.1141.2013.01033 虹鳟 LECT2 的酵母表达、纯化及生物活性分析 * 史雨红，章瑞程，杨旦阳，陆新江，陈 炯 ，李明云 宁波大学 应用海洋生物技术教育部重点实验室, 浙江 宁波 315211 摘要：白细胞衍生趋化因子2 (leukocyte cell-derived chemotaxin 2, LECT2)是一个参与多种生理和病理过程的分泌型细 胞因子。该文采用毕赤酵母表达体系分泌表达虹鳟LECT2 ，用阳离子交换柱结合分子筛层析方法分离纯化目的蛋白， 并获得纯度为96%，得率为120 mg/L 的重组虹鳟 (Oncorhynchus mykiss)LECT2 酵母培养物。生物学活性验证表明该重 组蛋白能趋化虹鳟头肾来源的巨噬细胞，增强其呼吸爆发和杀菌能力，并改变其细胞因子等基因的表达。综上，该实 验建立了一种快速有效的虹鳟LECT2 活性重组蛋白的制备方法，为后续相关蛋白的功能研究奠定了基础。 关键词：LECT2 ；毕赤酵母；纯化；生物活性；虹鳟 中图分类号：Q503; Q959.4 文献标志码：A 文章编号：0254-5853-(2013)01-0033-06 Yeast-based production, purification and bioactivity assay of rainbow trout LECT2 * Yu-Hong SHI, Rui-Cheng ZHANG, Dan-Yang YANG, Xin-Jiang LU, Jiong CHEN , Min-Yun LI Key laboratory of Applied Marine Biotechnology, Ministry of Education , Ningbo University, Ningbo 315211, China Abstract: Leukocyte cell-derived chemotaxin 2 (LECT2) is a secretory cytokine that functions in many physiological and pathological processes. We used a Pichia pastoris expression system for the recombinant expression of rainbow trout LECT2. The recombinant LECT2 was purified by UNOsphere S Cation exchange and size-exclusion chromatography columns. The obtained target protein was highly pure (96% homogeneity) and the yield was 120 mg/L of yeast cultures. An in vitro chamber assay revealed that recombinant LECT2 could induce chemotactic responses in rainbow trout head kidney-derived macrophages. Recombinant LECT2 not only enhanced macrophage respiratory burst activity and bactericidal activity, bu