Cep70的细胞定位分析及在微管聚合中的作用研究  

Study of cellular localization of Cep70 and its role in microtubule polymerization

作　　者：张俞 史兴娟 

机构地区：[1]东南大学生命科学研究院/发育与疾病相关基因教育部重点实验室,江苏南京210096

出　　处：《南京农业大学学报》2017年第6期1094-1099,共6页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(81770381;31301113);江苏省自然科学基金项目(BK20130607);中央高校基本科研业务费专项资金

摘　　要：[目的]本试验旨在研究Cep70的细胞定位及其在微管聚合中的调节作用。[方法]利用原核表达质粒p MALp2TCep70,在细菌中诱导表达融合蛋白MBP-Cep70。用融合蛋白免疫小鼠,获得特异性的抗Cep70抗体。首先用该抗体对细胞进行免疫荧光显微分析,检测Cep70在细胞内的定位,再利用荧光标记的微管体外组装试剂盒研究Cep70在体外微管聚合中对微管形态的调节,最后利用微管混浊度试验,验证Cep70对微管聚合的功能影响。[结果]通过从细菌中诱导纯化蛋白,获得了大量高纯度的融合蛋白MBP-Cep70。纯化蛋白免疫小鼠后得到了靶向Cep70的抗体。免疫荧光显微分析发现Cep70定位在微管组织中心(MTOC),即中心体上。通过对荧光标记的微管蛋白进行体外组装分析,发现Cep70增加了体外聚合微管的长度。进一步通过微管浑浊度试验,发现Cep70促进了体外微管的聚合。[结论]Cep70在细胞内定位在微管组织中心上,且在体外微管聚合上具有一定促进作用。[Objectives] The paper aims to study cellular localization of Cep70 and its role in mierotubule polymerization. [ Methods] Prokaryotic expression plasmid of pMALp2T-CepTO was constructed and transfected into BL21 strain of Escherichia coli. MBP-Cep70 fusion protein was induced and purified from bacteria. Anti-Cep70 antibody was obtained by immunizing mice with MBP-CepTO fusion protein. We first analyzed the cellular localization of Cep70 with anti-CepT0 antibody by immunofluorescence microscopy. We then analyzed the role of Cep70 in microtubule polymerization with in vitro tubulin polymerization assay by examining the morphology of rhodamine-labded tubulin. Finally,the role of Cep70 in microtubule assembly was further verified by measuring the absorbance of microtubule at a 350 nm wavelength at 37 ~C. [ Results ] We harvested large quantity of MBP-Cep70 fusion protein by purification from bacteria,and obtained anti-Cep70 antibody by immunizing mice with fusion protein. The cellular localization of Cep70 was examined with anti-CepT0 antibody by immunofiuorescence microscopy analysis. We found that Cep70 protein localized at the microtubule organizing center. In vitro microtubule polymerization assay demonstrated that Cep70 increased the length of polymerized microtubule. By measuring the absorbance of microtubule at 350 nm wavelength, we also found that Cep70 promoted in vitro microtuhule polymerization. [ Conclusions] Our results demonstrated that Cep70 localized at the microtubule organizing center, and promoted in vitro microtubule assembly.

关 键 词：Cep70 抗体 免疫荧光 微管聚合 

分 类 号：S852[农业科学—基础兽医学]