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

Prokaryotic Expression,Antibody Preparation of Gonad-specific Protein EsSOX21b-like of the Chinese Mitten Crab Eriocheir sinensis

Sox(SRY-related HMG-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Es Sox21b-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-Es Sox21b-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54 k D。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,Western blotting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28 k D,在精巢组织中的表达量比在卵巢中高,该结果暗示Es SOX21b-like可能在性腺发育过程中起重要调控作用。

The Sox（SRY-related HMG-box）is a large family of genes which encode transcription factors with high-mobility-group DNA binding domain related to the SRY（sex determining region Y）. In previous studies we isolated and identifiedEsSox21b-like transcripts exclusively expressed only in gonads of the Chinese mitten crab（Eriocheir sinensis）, this study is to verify whetherEsSOX21b-like protein is also specifically expressed in the gonad. GeneEsSox21b-like was cloned to expression vector pGEX-2T, the recombinant prokaryotic expression plasmid pGEX-2T-EsSox21b-like was constructed, then transferred into hostEscherichia coli BL21, and induced by IPTG. SDS-PAGE analysis revealed that the fusion protein was expressed as inclusion body with the molecular weight of 54 kD. The recombinant proteins were subsequently purified by Ni＋ affinity chromatography. Then the purified protein was used to immunize rabbits for preparation of theEsSOX21b-like antibody. Western blotting detection showed the antibody specifically recognized theEsSOX21b-like protein in the gonad, with molecular weight of 28 kD, whereas no expression was detected in other tissues. The expression level of the target protein was much higher in testis than that in ovary. This result implied that theEsSOX21b-like protein could have a regulatory role in the gonad development.