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Prokaryotic expression, antibody preparation, and identification of a cyclin B protein in the Chinese mitten crab Eriocheir sinensis and Malaysian giant prawn Macrobrachium rosenbergii
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Key Laboratory of Aquatic Genetic Resources and Utilization Certificated by Ministry of Agriculture, E-Institute of Shanghai Universities (EISU), Shanghai Ocean University

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Q959

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    Abstract:

    We amplified the open reading frames (ORFs) of the cyclin B gene from the ovarian cDNA of the Chinese mitten crab Macrobrachium rosenbergii. The ORFs from these two species were then ligated into the expression plasmids pGEX-2T and pET32a, respectively. Following transfer of the recombinant plasmids into a host bacteria, BL21 (DE3), the recombinant proteins were expressed as inclusion bodies by induction with IPTG. The optimum concentration of IPTG was 0.1 mmol/L for 4 h at 37C for GST-EsCB and 0.1 mmol/L PTG for 6 h at 30C for Trx-MrCB. The recombinant proteins were subsequently purified by affinity chromatography. The cyclin B antibody was further refined by immunizing New Zealand white rabbits with the purified protein. The highest resulting antiserum titer was 180 000 (ELISA). We verified the specificity of the antibodies against the recombinant protein and ovarian cyclin B protein by western blot. We isolated two isoforms of the cyclin B protein from the immature crab ovaries, but only one form from the prawn ovaries. Thus, our data suggest that the role of cyclin B in the regulation of oocyte maturation may differ between crabs and prawns. The cyclin B antibody will be useful for further study of the role of cyclin B on MPF activation and oocyte maturation.

    Reference

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        房君江中华绒螯蟹、罗氏沼虾基因克隆及其在卵子发生过程中表达分析上海上海海洋大学

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冯海洋,邱高峰. 中华绒螯蟹和罗氏沼虾cyclin B蛋白的原核表达、抗体制备及其鉴定[J]. Jounal of Fishery Sciences of China, 2011,[volume_no](4):713-719

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  • Online: August 17,2011
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