摘要
目的观察体外人类嗜T淋巴细胞病毒(HTLV-Ⅰ)Tax蛋白与Mller细胞蛋白结合及其靶蛋白组成成分。方法用含Tax蛋白cDNA的原核表达载体PGEX-3X-GST-Tax转化大肠杆菌,诱导表达并提取纯化GST-Tax融合蛋白;细胞外GST-Tax融合蛋白与Mller细胞蛋白结合,GS4B下拉(pull-down)与GST-Tax结合的蛋白;应用二维凝胶电泳、质谱分析(MALDI-Tof-MS)和免疫印迹分析等方法分析鉴定结合的蛋白。结果通过与对照组比较,在二维凝胶电泳上观察到多个差异蛋白质斑点,经质谱分析鉴定出10个差异蛋白质为Tax的候选靶蛋白,经免疫印迹,初步证实Tax蛋白能与ENO1相结合。结论初步分离鉴定出Mller细胞中的ENO1等蛋白质可能为Tax蛋白的靶蛋白分子。
Objective Human T-cell leukemia virus type 1 （HTLV- Ⅰ ） is associated with many ocular diseases. The viral transcriptional activator, Tax, has the primary role to directly promote viral mRNA synthesis in the viral life cycle and has numerous and crucial roles in the pathogenesis of HTLV- Ⅰ induced diseases. The current study aimed to investigate the target proteins of Tax in Müller cells in vitro. Methods An E. eoli strain, BL21DE3, was transfected with PGEX-3X-GST-Tax plasmid. The GST-Tax fusion protein expressed in E. coli. was separated by the centrifugation. The lysates of Mailer cells interacted with the GST-Tax fusion protein and then was purified by the GST pull down assay. The combined proteins of GST-Tax were separated and identified by the proteomics methods （ two dimensional gel electrophoresis and MALDI-TOF-MS）. One of identified protein,ENO1 ,was examined by pull down and Western blot. Results There were ten diverse proteins,including carbonyl reductase, tubulin, ATP synthase, Pdia3, laminin-binding protein, IGFBP-3, ENO1 and actin like protein, were found by the two dimentional gel electrophoresis and MALDI-TOF-MS. Western blotting with anti-ENO1 revealed that Tax could be binded to ENO1. Conclusion The results suggest that target proteins of Tax can be separated and identified in the lysates of Mailer cells. Tax can interact with ENO1 in vitro.
出处
《眼科研究》
CSCD
北大核心
2008年第9期689-692,共4页
Chinese Ophthalmic Research
基金
国家自然科学基金资助(30572002)
作者简介
通讯作者：吴开力（E-mail：wukaili@mail．sysu．edu．cn）
