State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen 361005, Fujian, China.
Fish Shellfish Immunol. 2012 Oct;33(4):725-35. doi: 10.1016/j.fsi.2012.06.011. Epub 2012 Jul 11.
In vertebrates, lymphoid-specific recombinase protein encoded by recombination-activating genes (RAG1/2) plays a key role in V(D)J recombination of the T-cell receptor and B-cell receptor. In this study, both RAG1 and the immunoglobulin M (IgM) mu chain were cloned to characterize their potential role in the immune defense at developmental stages of red-spotted grouper, Epinephelus akaara. The open reading frame (ORF) of E. akaara RAG1 included 2778 nucleotide residues encoding a putative protein of 925 amino acids, while the ORF of the IgM mu chain had 1734 nucleotide residues encoding 578 amino acids including variable (VH) and constant (CH1-CH2-CH3-CH4) regions. E. akaara RAG1 was composed of a zinc-binding dimerization domain (ZDD) with a RING finger and zinc finger A (ZFA) in the non-core region and a nonamer-binding region (NBR), with a zinc finger B (ZFB), the central and C-terminal domains in the core region. Tridimensional models of the ZDD and NBR of E. akaara RAG1 were constructed for the first time in fishes, while a 3D model of the E. akaara IgM mu chain was also clarified. The RAG1 mRNA was only detected in the thymus and kidney of 4-month and 1.5-year old groupers using qPCR, and the RAG1 protein was confirmed using western blotting and immunohistochemistry. The IgM mu mRNA was examined in most tissues except the gonad. RAG1 and IgM mu gene expression were observed at 15 dph (days post-hatching) and 23 dph respectively, and increased to a higher level at 37 dph. In addition, this was the first time that the morphology of the E. akaara thymus was characterized. The oval-shaped thymus of 4-month old fish was clearly seen and there were amounts of T lymphocytes present. The results suggested that the immune action of E. akaara was likely to start to develop around 15 dph to 29 dph. The transcript level of the RAG1 gene and the number of lymphocytes in the thymus between 4-month and 1.5-year old groupers indicated that age-related thymic atrophy also occurs in fishes. The similar functional structures of RAG1 and IgM protein between fish and mammals indicated that teleost species share a similar mechanism of V(D)J recombination with higher vertebrates.
在脊椎动物中，由重组激活基因（RAG1/2）编码的淋巴特异性重组酶蛋白在 T 细胞受体和 B 细胞受体的 V(D)J 重组中发挥关键作用。在这项研究中，我们克隆了 RAG1 和免疫球蛋白 M（IgM）μ链，以研究它们在红鳍石斑鱼（Epinephelus akaara）发育阶段的免疫防御中的潜在作用。E. akaara RAG1 的开放阅读框（ORF）包含 2778 个核苷酸残基，编码一个由 925 个氨基酸组成的假定蛋白，而 IgM μ链的 ORF 有 1734 个核苷酸残基，编码包含可变（VH）和恒定（CH1-CH2-CH3-CH4）区的 578 个氨基酸。E. akaara RAG1 由锌结合二聚化结构域（ZDD）组成，该结构域在非核心区域具有一个 RING 指和锌指 A（ZFA），以及一个非九聚体结合区（NBR），核心区域包含一个锌指 B（ZFB）、中央结构域和 C 末端结构域。这是首次在鱼类中构建了 E. akaara RAG1 的 ZDD 和 NBR 的三维模型，同时也阐明了 E. akaara IgM μ链的三维模型。使用 qPCR 仅在 4 月龄和 1.5 岁石斑鱼的胸腺和肾脏中检测到 RAG1 mRNA，并用 Western blot 和免疫组织化学法证实了 RAG1 蛋白的存在。在除性腺外的大多数组织中都检测到了 IgM μ mRNA。RAG1 和 IgM μ 基因的表达分别在 15 dph（孵化后第 15 天）和 23 dph 观察到，在 37 dph 时表达水平增加到更高水平。此外，这是首次对 E. akaara 胸腺的形态进行了描述。在 4 月龄鱼中可以清楚地看到椭圆形的胸腺，并且存在大量的 T 淋巴细胞。结果表明，E. akaara 的免疫作用可能在 15 dph 至 29 dph 左右开始发展。4 月龄和 1.5 岁石斑鱼之间 RAG1 基因的转录水平和胸腺中的淋巴细胞数量表明，鱼类也存在与年龄相关的胸腺萎缩。鱼类和哺乳动物的 RAG1 和 IgM 蛋白的类似功能结构表明，硬骨鱼类与高等脊椎动物具有相似的 V(D)J 重组机制。
