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中华绒螯蟹性腺特异表达蛋白EsSOX21b-like的原核表达与抗体制备

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.06.030, PP. 189-194

Keywords: 中华绒螯蟹,EsSOX21b-like蛋白,原核表达,抗体制备

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Abstract:

Sox(SRY-relatedHMG-box)基因编码一类重要的转录因子,其产物都具有一个HMG基序保守区。之前我们在中华绒螯蟹分离鉴定了一个只在性腺中特异表达转录本EsSox21b-like,为了验证EsSOX21b-like蛋白是否也在中华绒螯蟹性腺中特异表达,本研究原核表达了EsSOX21b-like蛋白,即将EsSox21b-like基因序列克隆到pGEX-2T表达载体,构建重组表达质粒pGEX-2T-EsSox21b-like,转化大肠杆菌BL21,IPTG诱导融合表达,经SDS-PAGE分析表明,融合蛋白主要以包涵体形式存在,分子量约为54kD。利用Ni柱亲和纯化融合蛋白后免疫家兔,制备了多克隆抗体,Westernblotting检测表明该抗体能特异地识别性腺组织中EsSOX21b-like蛋白,而在其他组织未检测到其表达,目的蛋白分子量约为28kD,在精巢组织中的表达量比在卵巢中高,该结果暗示EsSOX21b-like可能在性腺发育过程中起重要调控作用。

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