细胞质基质中macroH2A分子伴侣的筛选
投稿时间:2016-04-13  修订日期:2016-05-05  点此下载全文
引用本文:林娜,张田甜.细胞质基质中macroH2A分子伴侣的筛选[J].医学研究杂志,2016,45(11):35-39
DOI: 10.11969/j.issn.1673-548X.2016.11.010
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作者单位E-mail
林娜 100005 北京, 中国医学科学院基础医学研究所生化系  
张田甜 100005 北京, 中国医学科学院基础医学研究所生化系 ztt365@163.com 
基金项目:国家自然科学基金资助项目(31471209)
中文摘要:目的 筛查细胞质基质中组蛋白H2A变体macroH2A的分子伴侣,借此研究macroH2A在分子伴侣的协助下进入细胞核的分子机制。方法 建立在羧基末端表达融合有Flag标签的macroH2A-HCD(histone core domain)(a.a.21~161)的HeLa S3稳定细胞系。大规模悬浮培养细胞并利用低渗处理膨胀细胞、匀浆、离心,分离得到细胞质基质。透析后,通过免疫共沉淀技术获得macroH2A-HCD蛋白复合物,利用质谱技术鉴定macroH2A-HCD蛋白复合物的成分,分析质谱数据筛选macroH2A分子伴侣。结果 通过PCR扩增macroH2A-HCD DNA片段,并将之连入感染用载体pWPXL-C1-Flag。酶切、测序鉴定正确的pWPXL-macroH2A-HCD-C1-Flag质粒经包装转染HEK293T产生的慢病毒,对HeLa S3进行感染。通过Western blot法及免疫荧光实验,证明稳定表达羧基末端融合Flag标签的macroH2A-HCD的HeLa S3细胞系构建成功。悬浮培养后收集的细胞,经过低渗溶液处理后体积膨胀为原来体积的2倍,经匀浆、离心,匀浆液自上而下依次分为4层,分别为脂膜层、细胞质基质层、细胞器层、核层,最后获得纯净的细胞质基质。细胞质基质中加入Flag-M2珠子,经过免疫共沉淀实验得到蛋白复合物。经质谱分析后发现,复合物中含有macroH2A-HCD及多种已知的分子伴侣。结论 细胞质基质中的macroH2A可能在多种分子伴侣的协助下进入细胞核。
中文关键词:细胞质基质  macroH2A  分子伴侣  免疫共沉淀  入核
 
Screening the Chaperone of MacroH2A in Cytoplasmic Matrix
Abstract:Objective To screen the molecular chaperone of histone variant macroH2A in cytoplasmic matrix and study the mechanism of macroH2A imported into nucleus with the assistance of chaperone. Methods Stable HeLa S3 cell line that express macroH2A-HCD (histone core domain) (a.a. 21-161) that fuse a Flag tag to the carboxyl-terminal was established. Large scales of cells were cultured in suspension and swelled in hypotonic buffer. The cytoplasmic matrix was separated by homogenate and centrifugation. After dialysis, the macroH2A-HCD complex was purified by co-immunoprecipitation assay. The components of macroH2A-HCD complex were identified by mass spectrometry. The mass spectrometry data was analyzed to screen the chaperone of macroH2A. Results The DNA fragment of macroH2A-HCD was amplified by PCR and inserted into the vector pWPXL-C1-Flag. After restriction enzyme digestion and screening, the correct plasmid pWPXL-macroH2A-HCD-C1-Flag were transfected into HEK293T cells to produce the lentivirus particles for infecting HeLa S3 cells. The expression of Flag-tagged macroH2A-HCD was validated by Western Blot and immunofluorescence. The volume of suspension cultured cells were swollen to the twice volume in the hypotonic buffer. After homogenate and centrifugation, four layers were formed:lipid membrane layer, cytoplasmic matrix layer, cell-organ layer and nucleus layer. Flag-M2 beads were injected into the cytoplasmic matrix, and the protein complex was purified by co-immunoprecipitation assay. The bait Flag tagged macroH2A-HCD and several well-known chaperons were identified by mass spectrometry. Conclusion The macroH2A was imported into nucleus possibly with the assistance of multiple chaperone.
keywords:Cytoplasmic matrix  MacroH2A  Chaperone  Co-immunoprecipitation  Imported into nucleus
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