七鳃鳗Lja-SHP2分子鉴定、重组表达及免疫学研究

高等脊椎动物的蛋白酪氨酸磷酸酶SHP2(SH2 domain-containing protein-tyrosine phosphatase-2)由ptpn11基因编码,催化酪氨酸残基去磷酸化,与其他能催化酪氨酸磷酸化的蛋白酪氨酸激酶共同调节机体内多种信号通路的信号传导。以往研究表明,SHP2在高等脊椎动物T细胞和B细胞的激活与信号转导过程中起着重要作用。为了研究无颌类脊椎动物日本七鳃鳗(Lampetra japonica)中与SHP2同源的分子——Lja-SHP2在免疫应答反应中的作用,本研究通过PCR扩增获取其Lja-SHP2开放阅读框序列,并构建到原核表达载体pET-32a中,成功在大肠杆菌中实现重组蛋白表达并制备了其兔源多克隆抗体。用混合菌免疫刺激日本七鳃鳗后,通过实时荧光定量PCR和免疫印迹方法检测了Lja-SHP2在日本七鳃鳗免疫相关组织中mRNA和蛋白水平表达谱。结果显示,混合菌免疫刺激后,Lja-SHP2 mRNA和蛋白表达在外周血白细胞和髓样小体中无显著变化,而在鳃组织中显著性上调(P<0.05),说明Lja-SHP2在混合菌刺激后主要参与了鳃组织的免疫应答反应。为了进一步探究Lja-SHP2与淋巴细胞亚群免疫应答反应的相关性,本研究分别使用B细胞有丝分裂原脂多糖(lipopolysaccharide,LPS)和T细胞的有丝分裂原植物凝集素(phytohemagglutinin,PHA)免疫刺激日本七鳃鳗。经LPS免疫刺激后,与对照组相比,白细胞中Lja-SHP2蛋白表达显著上调,鳃组织和髓样小体没有显著性差异表达;但经PHA免疫刺激后,与对照组相比,白细胞、鳃组织和髓样小体3种组织中Lja-SHP2均有上调,尤其在白细胞中上调最为显著,大约是对照组的2.5倍,说明Lja-SHP2参与了日本七鳃鳗由PHA介导的免疫应答反应。由于PHA能刺激日本七鳃鳗鳃组织中VLRA+淋巴细胞的活化,这表明Lja-SHP2可能参与了PHA介导的VLRA+淋巴细胞亚群的免疫应答反应。上述研究结果为进一步探索Lja-SHP2在七鳃鳗免疫应答过程中的功能奠定了基础,也为揭示SHP2分子家族的系统发生及探索高等脊椎动物适应性免疫系统的早期发生及其进化历程提供一定的线索。

Identification,recombinant expression and immunological study of Lja-SHP2 in Lampetra japonica

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.