Li Xin, Qu Cheng-Ming, Han Ying-Lun, Liu Xin, Li Qing-Wei
Research Center of Lamprey, School of Life Sciences, Liaoning Normal University, Dalian 116029, China.
Research Center of Lamprey, School of Life Sciences, Liaoning Normal University, Dalian 116029, China; Collaborative Innovation Center of Seafood Deep Processing, Dalian Polytechnic University, Dalian 116034, China.
Yi Chuan. 2020 Feb 20;42(2):183-193. doi: 10.16288/j.yczz.19-260.
The protein tyrosine phosphatase SHP2 of higher vertebrates, encoded by ptpn11 gene, catalyzes the dephosphorylation of tyrosine phosphorylation site, and plays regulatory roles in various signaling pathways by cooperating with other protein tyrosine kinase. Previous studies have shown that SHP2 plays an important role in the activation and signal transduction of T and B cells in higher vertebrates. To study the role of a SHP2 homologous molecule of lampreys (Lja-SHP2) in immune response, we cloned and expressed the open reading frame sequence of Lja-SHP2 gene in prokaryotic expression vector pET-32a. The recombinant protein was successfully expressed in E. coli and the rabbit-derived polyclonal antibody was prepared. Lampetra japonica were immunized with mixed bacteria, and the mRNA and protein of Lja-SHP2 in immune-related cells and tissues were detected by real-time quantitative PCR and Western blotting after immunization. The Lja-SHP2 mRNA and protein were not significantly affected in leukocytes and supraneural myeloid bodies, but up-regulated significantly in gill tissues (P<0.05) after challenged by mixed bacteria, which indicated that Lja-SHP2 mainly participates in the immune response of gill tissues after mixed bacteria stimulation. To further investigate whether Lja-SHP2 level was affected in three lymphocyte subsets, the B-cell mitogen lipopolysaccharide (LPS) and T-cell mitogen phytohaemagglutinin (PHA) were employed to boost the immune response in L. japonica. LPS immune stimulation increased Lja-SHP2 in leucocytes significantly compared with the control group, and but had a marginal effect on Lja-SHP2 expression in gills and supraneural myeloid bodies. PHA immune stimulation could up-regulate Lja-SHP2 level in leukocytes, gill tissues and supraneural myeloid bodies. The change of Lja-SHP2 was especially dramatical in leukocytes, which was about 2.5 times higher than that in the control group, suggesting that Lja-SHP2 is involved in the lamprey immune response mediated by PHA. Consistent with the previous finding that PHA could induce the activation of VLRA lymphocytes, our results showed that Lja-SHP2 might be included in the immune response of VLRA lymphocytes mediated by PHA in gills. This research will benefit exploring the functions of Lja-SHP2 in the immune response of lamprey and will provide clues for understanding the phylogenesis of SHP2 molecular family, and its roles in the early occurrence and evolution of adaptive immune system in higher vertebrates.
高等脊椎动物的蛋白酪氨酸磷酸酶SHP2由ptpn11基因编码，催化酪氨酸磷酸化位点的去磷酸化，并通过与其他蛋白酪氨酸激酶协同作用在各种信号通路中发挥调节作用。先前的研究表明，SHP2在高等脊椎动物T细胞和B细胞的激活及信号转导中起重要作用。为研究七鳃鳗的SHP2同源分子（Lja-SHP2）在免疫反应中的作用，我们在原核表达载体pET-32a中克隆并表达了Lja-SHP2基因的开放阅读框序列。重组蛋白在大肠杆菌中成功表达，并制备了兔源多克隆抗体。用混合菌免疫日本七鳃鳗，免疫后通过实时定量PCR和蛋白质印迹法检测免疫相关细胞和组织中Lja-SHP2的mRNA和蛋白。混合菌攻击后，Lja-SHP2的mRNA和蛋白在白细胞和神经上髓体中无显著变化，但在鳃组织中显著上调（P<0.05），这表明Lja-SHP2主要参与混合菌刺激后鳃组织的免疫反应。为进一步研究Lja-SHP2水平在三种淋巴细胞亚群中是否受影响，使用B细胞有丝分裂原脂多糖（LPS）和T细胞有丝分裂原植物血凝素（PHA）来增强日本七鳃鳗的免疫反应。与对照组相比，LPS免疫刺激显著增加了白细胞中的Lja-SHP2，但对鳃和神经上髓体中Lja-SHP2的表达影响较小。PHA免疫刺激可上调白细胞、鳃组织和神经上髓体中Lja-SHP2的水平。Lja-SHP2在白细胞中的变化尤为显著，约为对照组的2.5倍，表明Lja-SHP2参与了PHA介导的七鳃鳗免疫反应。与先前PHA可诱导VLRA淋巴细胞激活的发现一致，我们的结果表明Lja-SHP2可能参与了PHA介导的鳃中VLRA淋巴细胞的免疫反应。本研究将有助于探索Lja-SHP2在七鳃鳗免疫反应中的功能，并为理解SHP2分子家族的系统发生及其在高等脊椎动物适应性免疫系统早期发生和进化中的作用提供线索。
