七鳃鳗血清中补体C3和VLRB介导的免疫防御

Immune Defense Mediated by Complement C3 and VLRB in Serum of Lamprey

日本七鳃鳗(Lampetra japonica)属最原始的无颌类脊椎动物,是研究免疫起源与进化的重要模式生物。七鳃鳗血清中C3分子(L-C3)是其含量最高的补体成分,在补体经典激活途径和旁路激活途径中均发挥重要作用。本文通过PCR扩增获取C3分子α-γ链的基因序列,构建到原核表达载体p ET-28a,成功在大肠杆菌中表达C3部分蛋白质并制备多克隆抗体。利用流式细胞术和激光共聚焦证明,L-C3分布在神经轴体的胞浆中。免疫共沉淀及细胞沉积结果显示,七鳃鳗血清中,VLRB与L-C3蛋白相互作用并共沉积于靶细胞膜表面。天然L-C3和VLRB清除实验,验证其在参与七鳃鳗血清杀伤靶细胞时发挥重要作用。本研究为七鳃鳗补体系统的研究奠定了基础,为七鳃鳗免疫起源与进化提供重要资料。

As the most original jawless vertebrate,Lampetra japonica（ lamprey） is an important model of the study of innate and adaptive immunity. The third component of complement of lamprey（ lamprey C3,L-C3,C3）,which is the most abundant complement protein in serum,plays an important role in the activation of complement classical and alternative pathways. In previous study,a C3 molecule was identified from c DNA library of the supraneural body in Lampetra japonica and named L-C3α-γ. The α-γ chain sequence of C3 molecule was amplified by PCR and constructed into prokaryotic expression vector p ET-28 a. The peptide of α-γ chain of C3 molecule was successfully expressed in E. coli and the polyclonal antibody was prepared. Flow cytometry and laser scanning confocal microscopy analysis revealed that L-C3 located in the cytoplasm of supraneural body cells. The results of coimmunoprecipitation and cell deposition showed that VLRB interacted with L-C3 protein and both codeposited on the membrane surface of the target cells. Results from depletion of native L-C3 and VLRB demonstrate that they play an important role in killing target cells. Our study provides evidences for the study the origin and evolution of immune system and complement system in Lampetra japonica.