凋亡蛋白质原核表达及纯化.doc

凋亡蛋白质原核表达及纯化摘要：目的构建来自鸡贫血病毒（CAV，chicken anaemia virus）VP3基因编码的凋亡蛋白质表达载体，在大肠杆菌中进行表达，并研究凋亡蛋白质的纯化和稳定性。方法PCR（polymerase chain reaction）获得正确编码VP3基因片断，构建表达质粒pET-28a-VP3在E. coli BL21(DE3)中表达。用Ni-NTA柱纯化可溶性目标蛋白质，并研究不同条件下凋亡蛋白质的稳定性。结果目标蛋白质在大肠杆菌中获得表达，纯化后蛋白质纯度高于90 %，卡拉胶可明显提高重组蛋白质可溶状态下的稳定性。结论成功获得高纯度VP3可溶性蛋白质，增加了目标产物的稳定性，为进一步研究该蛋白质的临床应用奠定基础。 关键词：凋亡蛋白质；原核表达；可溶性；纯化；稳定性 中图分类号：Q786文献标识码：A文章编号：1672-979X(2008)03-0007-05 Expression, Purification and Activity Detection in vitro of Apoptin FU Wen-bing1, SHI Xuan1, ZHAO Jian1*, FAN Li-qiang1, LI Su-xia, WANG Fu-jun2, SONG Yu-wen1, YUAN Qing-sheng1 (1. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; 2. Zhejiang Rishengchang Pharmaceutical Co., Ltd, Zhejiang 322100, China) Abstract ：Objective To construct an soluble expression system in E. coli for apoptin which was encoded by VP3 gene of chicken anemia virus (CAV), and study the purification and stabilization of apoptin. Methods The VP3 gene was amplified by PCR , then the segment was inserted into pET-28a (+) and the expression vector pET-28a-VP3 was constructed in E. coli BL21(DE3). The recombinant soluble protein was expressed in E. coli BL21(DE3)and purified by Ni-NTA column chromatography. The stability of apoptin was analyzed under different conditions. Results The target protein was expressed in E. coli BL21(DE3). The purity of apoptin was beyond 90 %after purification. The target protein was better stabilized by carrageenan in E.coli BL21(DE3). Conclusion Apoptin with high purity and stability can be successfully obtained, which do a base of the further study on clinic application of apoptin. Key words：apoptin; prokaryotic expression; solubility; purification; stability 凋亡蛋白质是鸡贫血病毒（CAV）的VP3基因编码的小分子蛋白质，由121个氨基酸组成，含有2个脯氨酸富含区和2个碱性区，近N端（33～44a）含有一个11个氨基酸组成的核运输序列（NES），靠近C端（84～90，112～118）含有2个核定位序列（NLS）。 Noteborn等[1]研究揭示，凋亡蛋白质能够诱导人的肝癌、淋巴瘤、白血病、乳腺癌、肺癌等多种癌细胞的凋亡，以及转化细胞的凋亡，但对未转化的人双倍体细胞无损伤[2]。凋亡蛋白质的