日本七鳃鳗PAK4参与LPS介导的VLRB类淋巴细胞亚群免疫应答反应

Lampetra japonica PAK4 participates in LPS-mediated immune response of VLRB+lymphocyte subset

p21活化蛋白激酶4(p21-activated protein kinase 4,PAK4)是一类丝氨酸/苏氨酸蛋白激酶,参与调控多种信号通路,在高等脊椎动物免疫应答中起重要作用。在低等脊椎动物日本七鳃鳗(Lampetra japonica)中发现一个PAK4同源基因(Lja-PAK4),其开放阅读框长度为2544 bp,编码一个含有848个氨基酸的蛋白质。该分子与高等脊椎动物PAK4序列比对一致性最高,且具有该蛋白家族保守的两个功能结构域。利用通过原核表达、纯化的重组Lja-PAK4蛋白为抗原,成功制备了大鼠抗Lja-PAK4的多克隆抗体。用混合菌刺激七鳃鳗,采用实时荧光定量PCR和免疫印迹方法发现,Lja-PAK4的mRNA和蛋白质在外周血淋巴细胞和髓样小体中均显著性上调表达。采用B细胞丝裂原LPS和T细胞丝裂原PHA分别对七鳃鳗进行刺激后发现,经LPS刺激后Lja-PAK4的mRNA和蛋白质在外周血淋巴细胞和髓样小体中呈现出显著性上调表达,而它们在鳃囊中的表达受到抑制。以上结果说明Lja-PAK4参与了LPS介导的七鳃鳗VLRB+淋巴细胞亚群的免疫应答反应。

P21 activated protein kinase 4(PAK4)is a serine/threonine protein kinase that participates in regulating multiple signaling pathways and plays an important role in the immune response of higher vertebrates.The PAK4 homologous gene was also found in the lower vertebrate Lampetra japonica.The open reading frame of the lamprey PAK4 gene(Lja-PAK4)is 2544 bp,encoding a protein containing 848 amino acids.Through homologous sequence alignment,it was found that it has the highest sequence identity with higher vertebrates PAK4 s and has two conserved functional domains of PAK protein family,indicating the existence of PAK4 homolog in lampreys.A polyclonal antibody against Lja-PAK4 in rats was successfully prepared using the recombinant Lja-PAK4 protein as the antigen which was expressed and purified through prokaryotic expression.After stimulation with mixed bacteria,the mRNA and protein of Lja-PAK4 were significantly upregulated in lamprey peripheral blood lymphocytes and supraneural myeloid bodies by real-time fluorescence quantitative PCR and immunoblotting methods,respectively.After stimulation with B cell mitogen LPS and T cell mitogen PHA,it was found that the mRNA and protein of Lja-PAK4 were significantly upregulated in peripheral blood lymphocytes and supraneural myeloid bodies after LPS stimulation,while their expression in the gills was inhibited.These results indicate that Lja-PAK4 is involved in the LPS mediated immune response of the VLRB+lymphocytes in lamprey subsets in Lampetra japonica.