【摘要】 为探索七鳃鳗(Lampetra japonica)与32 kDB淋巴细胞接头分子(B-cell adaptor molecule of 32 kDa, Bam32)同源的分子Lja-Bam32基因在其免疫应答反应中的作用，利用嵌套PCR方法成功克隆了Lja-Bam32基因开放阅读框的cDNA序列，包含747 bp核苷酸，编码含有249个氨基酸残基的蛋白质。通过多序列比对发现，Lja-Bam32具有该家族两个特征结构域Src同源区2和Pleckstrin同源结构域和两个保守的酪氨酸磷酸化位点。系统发育分析显示，Lja-Bam32与不同物种的Bam32分子位于同一个聚类群，以上结果表明，无颌类七鳃鳗中存在Bam32家族分子。然后通过构建表达载体对Lja-Bam32进行了原核表达，并利用纯化后的重组蛋白成功制备了兔抗Lja-Bam32的多克隆抗体。利用实时荧光定量PCR方法以及免疫印迹方法分别检测了日本七鳃鳗免疫相关组织中Lja-Bam32基因的mRNA及蛋白在混合菌免疫刺激前后的差异表达情况。结果表明，与未免疫组相比，经过混合菌[念珠菌(Candida albicaus)、大肠杆菌(Escherichia coli)和金黄葡萄珠菌(Staphylococcus cutetvs)]免疫刺激后，Lja-Bam32的mRNA转录表达量在外周血淋巴细胞、鳃囊和髓样小体中显著上调；而该基因的蛋白水平表达量只在淋巴细胞和髓样小体中显著上调，在其他组织中没有明显的变化。由于VLRB~+淋巴细胞主要在外周血淋巴细胞和髓样小体组织中分布，推测Lja-Bam32应该与七鳃鳗类B细胞(VLRB~+淋巴细胞)的免疫应答反应有关。研究结果为深入研究日本七鳃鳗VLRB~+淋巴细胞适应性免疫应答的信号转导分子机制提供了参考。更多还原

【Abstract】 B-cell adaptor molecule of 32 kDa(Bam32) is a kind of lymphocyte adaptor protein, which is mainly expressed in vertebrate B lymphocytes and plays an important role in signal transduction of B cells and maturation of affinity antibody in germinal center. So far, research on Bam32 gene has focused on mammals, and the presence and function of Bam32 in jawless vertebrates has not been reported. Therefore, we chose lamprey as the research object to discover and verify the existence and function of Bam32 gene, in an effort to provide some clues for in-depth understanding of the immune response mechanism of the adaptive immune system of jawless vertebrates. In the current study, Lampetra japonica was taken as the research object to explore the preliminary role of a Bam32 homolog in Lampetra japonica(Lja-Bam32) in its immune response. The cDNA sequence of Lja-Bam32 open reading frame(ORF) was obtained by nested PCR with designed primers. The ORF region of Lja-Bam32 contained 747 bp nucleotides, encoding a protein containing 249 amino acid residues. According to multiple sequence alignment, Lja-Bam32 had two typical characteristic domains [Src homology 2(SH2), Pleckstrin homology(PH) domains] and two conserved tyrosine phosphorylation sites of Bam32 family. The SH2 domain specifically binded to phosphorylated tyrosine-rich upstream signaling protein molecules, while the PH domain had a strong molecular affinity for phosphoinositols. The phylogenetic analysis revealed that Lja-Bam32 was grouped in the same cluster with other Bam32 molecules identified from invertebrates to vertebrates. The prokaryotic expression of Lja-Bam32 was realized by constructing the expression vector, and the rabbit polyclonal antibody against Lja-Bam32 was successfully prepared with purified Lja-Bam32 recombinant protein. The expression levels of Lja-Bam32 mRNA and protein in Lampetra japonica immune-related tissues were detected by real-time fluorescence quantitative PCR and western blotting, respectively. The results showed that, compared with the control group, the mRNA transcription level of Lja-Bam32 significantly increased in peripheral blood lymphocytes, gills and supraneural myeloid bodies after immune stimulation. The expression levels of Lja-Bam32 significantly increased in lymphocytes and supraneural myeloid bodies after mixed antigens stimulation, but there was no obvious change in other tissues. The adaptive immune system of Lampetra japonica was mediated by VLRA, VLRB, and VLRC. VLRs could recognize a variety of antigens, lymphocytes specifically expressing VLRA, VLRC and VLRB had similar functions to T cells and B cells, respectively, Lampetra japonica T cells(VLRA~+, VLRC~+ lymphocytes) developed in thymus-like tissue in gills, and B-like cells(VLRB~+ lymphocytes) matured in medullary bodies. As VLRB~+ lymphocytes were mainly distributed in peripheral blood lymphocytes and supraneural myeloid bodies, we deduced that Lja-Bam32 was the immune response of VLRB~+ lymphocytes against bacterial infection. The results of this study lay a foundation for further study on the signal transduction molecular mechanism of VLRB~+ lymphocyte adaptive immune response in Lampetra japonica, it also provides the possibility for the subsequent in-depth study of Lja-Bam32.更多还原