采用RT-PCR方法扩增、克隆和鉴定了泥蚶(Tegillarca granosa)的金属硫蛋白(Metallothionein,MT)基因(TgMT)的开放阅读框(ORF).基于金属硫蛋白氨基酸序列构建的系统进化树表明,泥蚶和魁蚶(Scapharca broughtonii)的亲缘关系最近.利用ORF序列,构建原核重组表达质粒.重组质粒经PCR、酶切鉴定及测序验证后转化大肠杆菌(Escherichia coli) BL21(DE3),经IPTG诱导,表达重组蛋白,重组蛋白的分子量约为28.3 kD.可溶性分析表明重组蛋白主要以可溶性形式存在.用His-tag柱亲和纯化重组蛋白,并利用重组蛋白制备了兔抗血清.Real-time PCR和Western blot实验表明,金属硫蛋白在泥蚶的各个组织中都有表达,并且在消化腺中的表达量最高.采用激光共聚焦显微镜,对泥蚶的消化腺进行免疫组化定位分析,发现泥蚶的MT主要存在于消化腺腺管上皮细胞的胞质中.泥蚶MT的克隆和表达研究为进一步研究该蛋白在环境监测中的作用、探讨细胞解毒的分子生物学机制奠定了基础.%Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins. MTs are thought to be involved in the cellular detoxification of metals (e.g., Cd and Hg) and homeostasis of essential metal ions (e.g., Zn and Cu) in mammals. However, little is known about the functions of MT in bivalves. We cloned the cDNA encoding metallothionein of Tegillarca granosa (TgMT) using RT-PCR. The open reading frame (ORF) of TgMT was 234 bp encoding a polypeptide of 77 amino acids with a predicted molecular mass of 7.9 kD. Phy-logenetic analysis suggested that TgMT was most closely related to MT from Scapharca broughtonii. We constructed a recombinant expression plasmid (pET32a-MT) by inserting the TgMT ORF into the prokaryotic expression vector pET-32a. The recombinant TgMT was successfully expressed in E. Coli BL21 (DE3) following induction with IPTG SDS-PAGE analysis confirmed the expression of TgMT, which had a molecular mass of about 28.3 kD, in agreement with the expected molecular weight. The recombinant protein was primarily expressed as a soluble protein and was purified by Ni-NTA His-Bind Resin. We injected the purified TgMT into rabbits to obtain polyclonal antiserum against TgMT for use in immunoblotting experiments. Real time PCR and western blot analysis revealed that TgMT was distributed ubiquitously in a range of tissues. Expression was highest in the digestive glands of T. Granosa. We performed immunohistochemistry using a laser confocal microscope to examine the cell distribution of TgMT in the digestive gland. TgMT was primarily localized in the cytoplast of the digestive tubule epithelium. Our results provide insight into the utility of using MT proteins for environmental monitoring and into the mechanisms controlling detoxification in T. Granosa.
展开▼
