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

doi:10.13560/j.cnki.biotech.bull.1985.2015.06.030

Abstract

Abstract: 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 identified EsSox21b-like transcripts exclusively expressed only in gonads of the Chinese mitten crab(Eriocheir sinensis), this study is to verify whether EsSOX21b-like protein is also specifically expressed in the gonad. Gene EsSox21b-like was cloned to expression vector pGEX-2T, the recombinant prokaryotic expression plasmid pGEX-2T-EsSox21b-like was constructed, then transferred into host Escherichia 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 the EsSOX21b-like antibody. Western blotting detection showed the antibody specifically recognized the EsSOX21b-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 the EsSOX21b-like protein could have a regulatory role in the gonad development.

Key words: Chinese mitten crab(Eriocheir sinensis), EsSOX21b-like protein, prokaryotic expression, antibody preparation

Yang Guocui, Cui Zheng, Qiu Gaofeng. Prokaryotic Expression，Antibody Preparation of Gonad-specific Protein EsSOX21b-like of the Chinese Mitten Crab Eriocheir sinensis[J]. Biotechnology Bulletin, 2015, 31(6): 189-194.

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