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

Prokaryotic expression,antibody preparation,and identification of a cyclin B protein in the Chinese mitten crab Eriocheir sinensis and Malaysian giant prawn Macrobrachium rosenbergii

从中华绒螯蟹(Eriocheir sinensis)和罗氏沼虾(Macrobrachium rosenbergii)卵巢cDNA中克隆了cyclin B基因的开放阅读框,分别连接到pGEX-2T和pET-32a表达载体,转化大肠杆菌BL21(DE3),经IPTG诱导表达重组蛋白GST-EsCB和Trx-MrCB。通过优化培养温度、IPTG浓度以及诱导时间,得出重组蛋白的最佳表达条件分别为:0.1 mmol/L IPTG、37℃、4 h和0.1 mmol/L IPTG、30℃、6 h。重组蛋白主要以包涵体形式存在。亲和层析纯化重组蛋白,作为抗原免疫家兔,制备GST-EsCB和Trx-MrCB多克隆抗体。ELISA检测抗体效价均高达1∶80 000,Western blot鉴定该抗体能特异性识别重组蛋白,而且均能够从卵巢总蛋白中检测到cyclin B蛋白。在中华绒螯蟹未成熟卵巢中cyclin B蛋白出现2种亚型,在罗氏沼虾未成熟卵巢中只有1种亚型,推测虾和蟹cyclin B蛋白调控卵母细胞成熟的分子机制不同。本研究制备了cyclin B抗体,旨为在蛋白水平研究cyclin B在卵母细胞成熟过程中的作用机制提供基础条件。

We amplified the open reading frames（ORFs） of the cyclin B gene from the ovarian cDNA of the Chi-nese mitten crab Eriocheir sinensis and the Malaysian giant prawn 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,Escherichia coli 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 37？C for GST-EsCB and 0.1 mmol/L PTG for 6 h at 30？C 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 1∶80 000（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.