细胞质基质中macroH2A分子伴侣的筛选  

Screening the Chaperone of MacroH2A in Cytoplasmic Matrix

作　　者：林娜[1] 张田甜[1] 

机构地区：[1]中国医学科学院基础医学研究所生化系,北京100005

出　　处：《医学研究杂志》2016年第11期35-39,共5页Journal of Medical Research

基　　金：国家自然科学基金资助项目(31471209)

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HeLas3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-HCDDNA片段，并将之连人感染用载体pWPXL—C1-Flag。酶切、测序鉴定正确的pWPXL-macroH2A-HCD-Cl-Flag质粒经包装转染HEK293T产生的慢病毒，对HeLas3进行感染。通过Westernblot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HeLas3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。Objective To screen the molecular chaperone of histone variant macroH2A in cytoplasmic matrix and study the mecha- nism 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 earboxyl - terminal was established. Large scales of ceils were cultured in suspension and swelled in hypotonie buffer. The cytoplasmic matrix was separated by homogenate and centrifugation. After di- alysis, the maeroH2A - HCD complex was purified by co - immunopreeipitation 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 - Cl - Flag. After restriction enzyme di- gestion and screening, the correct plasmid pWPXL- macroH2A -HCD -C1 -Flag were transfected into HEK293T cells to produce the lentivirus particles for infecting HeLa $3 cells. The expression of Flag - tagged macroH2A - HCD was validated by Western Blot and im- munofluorescence. The volume of suspension cultured ceils 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 im- ported into nucleus possibly with the assistance of multiple chaperone.

关 键 词：细胞质基质 macroH2A 分子伴侣 免疫共沉淀 人核 

分 类 号：R3[医药卫生—基础医学]