小鼠干扰素-γ诱生单核因子在昆虫细胞中的高效表达及其血管生成抑制作用  

Large Scale Production and Purification of Murine Chemokine Monokine Induced by IFN-gamma (MIG, CXCL9) in Insect Cells and Its Angiostatic Effects

作　　者：张德新[1] 吴静[1] 杨静华 REUVE NRABINOVICI 樊代明[1] 

机构地区：[1]第四军医大学全军消化疾病研究所,西安710032 [2]Departments of Surgery,Yale School of Medicine,New Haven,Connecticut

出　　处：《生物化学与生物物理进展》2005年第10期975-981,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(39800156;30371337)~~

摘　　要：干扰素-γ诱生单核因子(monokineinducedbyinterferon-γ,MIG,CXCL,9)是一种具有趋化T淋巴细胞NK细胞的CXC趋化因子家族成员之一.通过RT-PCR技术从小鼠脑组织中克隆了小鼠MIGcDNA,利用Bac-to-Bac杆状病毒表达系统构建了MIG重组杆状病毒,并在Tn-581-4昆虫细胞内将其进行了表达,最后对表达产物进行了纯化和活性鉴定.结果显示:小鼠MIG蛋白在Bac-to-Bac杆状病毒表达系统中可得到高效分泌性表达;分泌到培养上清中的重组MIG蛋白经S-Sepharose纯化后得到的MIG蛋白纯度约为90%,产量为10mg/L;在SDS-聚丙烯酰胺凝胶电泳和蛋白质印迹分析中该蛋白质分子质量显示为14.4ku左右;该纯化蛋白在体外具有诱导CTLL-2淋巴细胞钙离子内流和抑制血管内皮细胞迁移和增值的活性,在鸡胚尿囊膜实验中小鼠重组MIG具有抑制新血管生成的功能.实验表明,在杆状病毒系统中高效表达的小鼠重组MIG蛋白具有抑制内皮细胞迁移、增殖和血管生成活性,为今后进一步研究其血管生成抑制活性的机理和将其应用于肿瘤血管抑制实验建立了基础.Monokine induced by interferon-γ （MIG, CXCL9） is a CXC chemokine which chemoattracts lymphocytes or neutrophils. Recent studies suggest that some members of CXC family, such as PF-4 and IL-8, CRG-2 and MIG also possess angiogenic or angiostatic properties. Recombinant mouse MIG protein were produced and purified and test its biological effects on endothelial cells. Mouse MIG cDNA was PCR amplified from mouse brain and cloned into pFASTBAC 1 vector in Bac-To-Bac baculovirus expression system. The recombinant mouse MIG Bacmid was introduced into High-Five （Tn-5B1-4） insect cells. After 48-72 h of incubation, the supernatant was collected and the MIG protein was purified by S-Sepharose. The yield of MIG was 10 mg/L and the purity is 90%. The MIG protein migrated as several bands on SDS-PAGE with the main band at 14.4 ku and was further confirmed by Western blot analysis. Functional assays demonstrated that purified mouse MIG protein not only elicited influx of calcium in mouse cytotoxic T cell lines but also inhibited both mouse endothelial cell migration and proliferation in a dose dependent manner. Thus, mouse MIG protein is not only a potent chemoattrant for lymphocytes but also an anti-angiogenic factor, which has important implications for angiogenesis-related diseases, such as tumor. It is the highest yield of recombinant mouse protein production described in the literature so far. The availability of large amount of purified active mouse MIG protein produced from baculovirus expression system has paved the way for the further studies of its in vivo angiostatic functions in mice.

关 键 词：CXC趋化因子 干扰素-γ诱生单核因子 杆状病毒 重组蛋白 血管生成 

分 类 号：Q718[生物学—分子生物学]