[1]张俞,史兴娟.Cep70的细胞定位分析及在微管聚合中的作用研究[J].南京农业大学学报,2017,40(6):1094-1099.[doi:10.7685/jnau.201707012]
 ZHANG Yu,SHI Xingjuan.Study of cellular localization of Cep70 and its role in microtubule polymerization[J].Journal of Nanjing Agricultural University,2017,40(6):1094-1099.[doi:10.7685/jnau.201707012]
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Cep70的细胞定位分析及在微管聚合中的作用研究()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
40卷
期数:
2017年6期
页码:
1094-1099
栏目:
出版日期:
2017-11-10

文章信息/Info

Title:
Study of cellular localization of Cep70 and its role in microtubule polymerization
作者:
张俞 史兴娟
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
Author(s):
ZHANG Yu SHI Xingjuan
Institute of Life Sciences/Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Southeast University, Nanjing 210096, China
关键词:
Cep70抗体免疫荧光微管聚合
Keywords:
Cep70antibodyimmunofluorescencemicrotubule polymerization
分类号:
S852
DOI:
10.7685/jnau.201707012
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pMALp2T-Cep70,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。
Abstract:
[Objectives] The paper aims to study cellular localization of Cep70 and its role in microtubule polymerization.[Methods] Prokaryotic expression plasmid of pMALp2T-Cep70 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-Cep70 fusion protein. We first analyzed the cellular localization of Cep70 with anti-Cep70 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-labeled 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℃.[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-Cep70 antibody by immunofluorescence 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 microtubule polymerization.[Conclusions] Our results demonstrated that Cep70 localized at the microtubule organizing center, and promoted in vitro microtubule assembly.

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备注/Memo

备注/Memo:
收稿日期:2017-07-06。
基金项目: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
作者简介:张俞,硕士,助理工程师。
通信作者:史兴娟,博士,讲师,主要从事心血管疾病与细胞骨架方面的研究,E-mail:xingjuanshi@seu.edu.cn。
更新日期/Last Update: 1900-01-01