斑马鱼Fbxl5基因多克隆抗体的制备与检测

盛力翔; 冯德峰; 罗世锋; 吴秀山; 戴国; 莫小阳

doi:doi:10.3969/j.issn.1007-7146.2013.03.010

激光生物学报, 2013, 22 (3): 249, 网络出版: 2015-07-24

斑马鱼Fbxl5基因多克隆抗体的制备与检测

Preparation and Detection of Fbxl5 Fusion Protein and Preparation of Its Polyclonal Antibody

论文大纲

盛力翔 ^*冯德峰罗世锋吴秀山戴国莫小阳

作者单位

湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室, 心脏发育研究中心, 湖南长沙 410081

融合蛋白多克隆抗体基因芯片 Fbxl5 Fbxl5 fusion protein polyclonal antibody gene chip

摘要

Fbxl5为F-box基因家族的一员, 目前研究发现其与心脏的发育有关。为了在斑马鱼模型中进一步研究该基因的功能, 有必要制备其多克隆抗体。通过桥式PCR扩增出亲水性和特异性均好的斑马鱼Fbxl5基因片段, 将其克隆入表达载体pET-28a, 转化至大肠杆菌Rosseta中。用IPTG诱导Fbxl5重组质粒得到His-Fbxl5的融合蛋白。这个融合蛋白用Ni-IDA凝胶柱亲和纯化, 将纯化的His-Fbxl5融合蛋白免疫新西兰大白兔制备多克隆抗体。使用Western Blot检测, 获得了Fbxl5原核表达重组融合蛋白及高效价的特异性兔抗Fbxl5多克隆抗体。随后检测了Fbxl5在斑马鱼胚胎和成体组织中蛋白的表达, 通过基因芯片分析在Fbxl5-MO和Std胚胎样品的mRNA含量差异.所得结果显示获得了较高效价和特异性好的斑马鱼Fbxl5多克隆抗体, 为Fbxl5功能的进一步研究奠定了基础。

Abstract

As a member of F-box gene family, Fbxl5 has been involved in the development of heart by the current research. In order to study the further function of Fbxl5 gene in Zebrafish model, it is necessary to prepare its polyclonal antibody. The hydrophilic and well specific fragment of Zebrafish Fbxl5 was obtained by Bridge PCR amplifications, then it was cloned into the expression vector pET-28a and transformed into Escherichia coli Rosseta. Fbxl5 recombinant plasmid was induced with IPTG to acquire His-Fbxl5 fusion protein. The induced protein was purified by Ni-IDA gel column, then it was immuned into the New Zealand white rabbit. The antibody’s titer and specification was identified by Western Blot separately.We also examined the expression of Fbxl5 in embryo and tissues of adult zebrafish,analyzed the content difference of mRNA between Fbxl5-MO and Std of the sample embryos in zebrafish by gene chip. And all the results showed that high sensitivity and specificity anti-Fbxl5 polyclonal antibody was generated, which provided a powerful tool for the further studies on Fbxl5.

参考文献

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盛力翔, 冯德峰, 罗世锋, 吴秀山, 戴国, 莫小阳. 斑马鱼Fbxl5基因多克隆抗体的制备与检测[J]. 激光生物学报, 2013, 22(3): 249. SHENG lixiang, FENG defeng, LUO shifeng, WU Xiushan, DAI Guo, MO Xiaoyang. Preparation and Detection of Fbxl5 Fusion Protein and Preparation of Its Polyclonal Antibody[J]. Acta Laser Biology Sinica, 2013, 22(3): 249.

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