Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China.
Laboratory of Pathology and Immunology of Aquatic Animals, KLMME, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266071, China.
Fish Shellfish Immunol. 2019 Oct;93:641-651. doi: 10.1016/j.fsi.2019.07.059. Epub 2019 Jul 22.
Interleukin-2 receptor subunit beta of flounder (Paralichthys olivace, fIL-2Rβ) was annotated on the NCBI, its gene was cloned and characterized functionally in this study. And then the amino acids sequences and tertiary structure of fIL-2Rβ were analyzed, respectively. RT-PCR and ImageJ analyzed showed that fIL-2Rβ mRNA were expressed in the gill, spleen, kidney, intestines, liver, blood, muscle and skin, which showed high signals in spleen and blood. And then the recombinant protein of fIL-2Rβ extracellular region and its polyclonal antibodies were produced, native fIL-2Rβ molecules in flounder peripheral blood leukocytes (PBLs) were identified at 60.7 kDa by Mass spectrometry, which were in accordance with the molecular mass of full fIL-2Rβ protein calculated on the predicted protein sequence. Then the IL-2Rβ+ cell in T/B lymphocytes were characterized by Flow cytometry and indirect immunofluorescence assay, respectively. The results showed that the percentages of IL-2Rβ+ leukocytes, IL-2Rβ+/CD4+, IL-2Rβ+/IgM+ lymphocytes were 18.4 ± 2.7%, 4.5 ± 0.8%, 4.3% ± 0.5 in PBLs, and were 13.6 ± 0.9%, 4.6 ± 1.1%, 6.1% ± 0.4 in spleen, similarly, the percentages of IL-2Rβ+ leukocytes, IL-2Rβ+/CD4+, IL-2Rβ+/IgM+ lymphocytes were 9.4 ± 0.3%, 4.0 ± 0.5%, 5.7 ± 0.1% in head kidney, respectively. After KLH injection, compared with control group, the gene expression of IL-2, IL-2Rβ, CD3, TCR, CD79b and IgM in spleen of flounder were up-regulated, respectively (p < 0.05). And the FCM results showed that the percentages of IL-2Rβ+ leukocytes in PBLs were significantly increased post Keyhole limpet hemocyanin (KLH) injection, which peaked 23.9 ± 0.9% at 9 day (p < 0.05). To our knowledge, those results first reported that the characteristics of IL-2R and IL-2R + molecules were expressed on both B and T lymphocytes in fish. At the same time, this study lays a foundation for further exploring the interaction between IL-2 and IL-2R to promote cell proliferation and carrying out biological functions.
牙鲆白细胞介素 2 受体亚基β（Paralichthys olivace，fIL-2Rβ）在 NCBI 上进行了注释，本研究对其基因进行了克隆和功能表征。然后分别对 fIL-2Rβ的氨基酸序列和三级结构进行了分析。RT-PCR 和 ImageJ 分析显示，fIL-2Rβ mRNA 在牙鲆的鳃、脾、肾、肠、肝、血、肌肉和皮肤中表达，在脾和血中呈现高信号。然后生产了 fIL-2Rβ 细胞外区的重组蛋白及其多克隆抗体，通过质谱法鉴定了牙鲆外周血白细胞（PBLs）中天然 fIL-2Rβ 分子，其分子量与预测的全长 fIL-2Rβ 蛋白的分子量一致。然后通过流式细胞术和间接免疫荧光法分别对 T/B 淋巴细胞中的 IL-2Rβ+细胞进行了鉴定。结果表明，PBLs 中 IL-2Rβ+白细胞、IL-2Rβ+/CD4+、IL-2Rβ+/IgM+淋巴细胞的百分比分别为 18.4±2.7%、4.5±0.8%、4.3%±0.5%，脾中分别为 13.6±0.9%、4.6±1.1%、6.1%±0.4%，同样，头肾中 IL-2Rβ+白细胞、IL-2Rβ+/CD4+、IL-2Rβ+/IgM+淋巴细胞的百分比分别为 9.4±0.3%、4.0±0.5%、5.7±0.1%。KLH 注射后，与对照组相比，牙鲆脾中 IL-2、IL-2Rβ、CD3、TCR、CD79b 和 IgM 的基因表达均上调（p<0.05）。FCM 结果表明，PBLs 中 IL-2Rβ+白细胞的百分比在注射钥孔血蓝蛋白（KLH）后显著增加，在第 9 天达到峰值 23.9±0.9%（p<0.05）。据我们所知，这些结果首次报道了鱼类 B 和 T 淋巴细胞上均表达 IL-2R 和 IL-2R+分子的特征。同时，本研究为进一步探索 IL-2 与 IL-2R 之间的相互作用，促进细胞增殖和发挥生物学功能奠定了基础。
