文蛤(Meretrix meretrix)铁蛋白的重组表达及其组织表达特征分析

构建了文蛤铁蛋白的重组表达质粒 pGEX-4T-1-MmeFer, 在大肠杆菌 Escherichia coli 中获得了重组蛋白, 经酶解和质谱鉴定该蛋白为重组文蛤铁蛋白(rGST-MmeFer), 切除 GST 标签的重组文蛤铁蛋白(rMmeFer)具有氧化和吸收铁离子的功能。利用 rGST-MmeFer 制备了兔抗文蛤铁蛋白的多克隆抗体, 进而利用 Western blot 的方法对文蛤铁蛋白的组织表达分布进行了研究。结果表明, 铁蛋白在文蛤各组织中均有分布, 其中消化腺中表达量最高, 足中表达量最低。此外, 利用实时定量PCR 分析了文蛤消化腺、外套膜和鳃组织中铁蛋白 mRNA 的表达, 发现铁蛋白 mRNA 在外套膜中表达量最高, 消化腺次之, 鳃中表达量最低, 差异达到显著水平(P<0.05)。上述结果为进一步研究铁蛋白参与贝类贝壳形成的分子机制奠定了基础。

CHARACTERIZATION OF RECOMBINANT EXPRESSION AND TISSUE EXPRESSION OF FERRITIN IN CLAM MERETRIX MERETRIX

Ferritin is involved in biomineralization of mollusk shells, although the molecular mechanism of this process is not fully understood. High level of recombinant protein rGST-MmeFer in Meretrix meretrix was obtained using Escherichia coli and then identified using in-gel digestion and LC-ESI-MS/MS. After rGST-MmeFer was purified on the GST affinity column and then the GST tag was cleaved, the remaining recombinant MmeFer (rMmeFer) exhibited similar function of iron oxidation and uptake in vitro. Rabbit anti-rGST-MmeFer polyclonal antibodies was prepared using the purified rGST-MmeFer and then used in Western blot analysis. The result showed that ferritin was expressed in all tissues of M. meretrix, with the highest expression in the digestive gland and the lowest in the foot. mRNA expression of the M. meretrix ferritin showed the highest expression in the mantle, followed by the digestive gland, and the lowest in the gill. Significant expression variation was found between the tissues (P<0.05). The results laid foundation for the functional analysis of ferritin involved in the shell formation of mollusks.