南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (02): 196-200.

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巢蛋白的克隆、表达、抗体制备及免疫组化分析

陈新林； 刘勇； 肖新莉； 张瑾； 吕海侠； 张蓬勃； 刘建新； 赵建军；   

  1. 西安交通大学医学院神经科学研究中心； 西安交通大学医学院第二附属医院； 西安交通大学医学院神经科学研究中心 西安交通大学医学院环境与疾病相关基因教育部重点实验室陕西西安710061； 西安交通大学医学院环境与疾病相关基因教育部重点实验室陕西西安710061； 陕西西安710004；
  • 出版日期:2006-02-20 发布日期:2006-02-20
  • 基金资助:
    国家自然科学基金(30300109,30170300)~~

Cloning, expression, and antibody preparation of nestin with immunohistochemical analysis

CHEN Xin-lin, LIU Yong, XIAO Xin-li, ZHANG Jin, L(?) Hai-xia, ZHANG Peng-bo, LIU Jian-xin, ZHAO Jian-jun Research Center for Neuroscience, Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, Medical School of Xi’an Jiaotong University, Xi’an 710061, China; Second Hospital, Medical School of Xi’an Jiaotong University, Xi’an 710004, China   

  1. 西安交通大学医学院神经科学研究中心； 西安交通大学医学院第二附属医院； 西安交通大学医学院神经科学研究中心 西安交通大学医学院环境与疾病相关基因教育部重点实验室陕西西安710061； 西安交通大学医学院环境与疾病相关基因教育部重点实验室陕西西安710061； 陕西西安710004；
  • Online:2006-02-20 Published:2006-02-20

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摘要： 目的在原核系统中克隆、表达并纯化巢蛋白(nestin),制备特异性nestin抗体,用于研究nestin在中枢神经系统发育中的生物学特性,探索神经发育及再生规律。方法采用RT-PCR方法由人神经干细胞中获取nestin cDNA,与原核表达载体pQE30连接,构建重组载体pQE30-nestin。测序后,将重组质粒转入大肠杆菌M15,IPTG诱导表达His融合蛋白。Ni-NTA亲和层析柱纯化后,SDS-PAGE电泳和Western blotting鉴定。用此蛋白免疫BALB／c小鼠,获得抗血清。 Western blotting、ELISA和免疫组织化学分析鉴定获取的抗血清。结果成功由人神经干细胞中克隆nestin cDNA片段。 IPTG诱导重组质粒pQE30-nestin表达出一相对分子质量约25 000的His融合蛋白并被Ni-NTA成功纯化,Western blotting证明其确为nestin蛋白。动物免疫后,经Western blotting、ELISA和免疫组化鉴定,获得的抗血清可特异性结合重组nestin蛋白、发育期人及大鼠脑内的nestm蛋白。结论获得了重组人nestin蛋白,制备的抗血清不仅可识别重组 nestin蛋白,亦可识别人及大鼠脑组织内的nestin蛋白。

Abstract: Objective To obtain recombinant nestin and prepare anti-nestin polyclonal antibody (mAb) to explore the biological roles of nestin in the central nervous system development. Methods The nestin cDNA was cloned from human neural stem cells by RT-PCR and ligated to prokaryotic expression plasmid pQE30 for construction of the recombinant vector pQE30-nestin. After sequencing, the recombinant vector was transformed into E.coli M15 and His-tagged nestin was induced by IPTG. The nestin was purified by Ni-NTA affinity chromatography column and characterized by SDS-PAGE and Western blotting. BALB/c mice were immunized with the purified recombinant protein to prepare the antiserum, which was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. Results The nestin gene was successfully cloned from human neural stem cells, which was identical to that reported in GenBank. After IPTG induction, the E.coli transformed with pQE30-nestin plasmid expressed a 25 000 His-tagged protein, which was successfully purified and i-dentified as nestin by Western blotting. Western blotting, ELISA and immunohistochemistry demonstrated that the antiserum could specifically bind to the recombinant nestin as well as to nestin in fetal human and rat brains. Conclusion We successfully cloned the nestin gene and expressed the nestin, and nestin mAb prepared can specifically recognize not only the recombinant nestin, but also nestin from human and rats brain tissues. 

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