中华鳖趋化因子CXCL10的分子特征及其对细菌感染的响应

Molecular characterization of CXCL10 in Chinese soft-shelled turtle (Pelodiscus sinensis) and its response to bacterial infection

摘要:

为研究趋化因子基因cxcl10的表达特征及其对外源病原微生物的响应，实验以中华鳖为对象，利用生物信息学分析CXCL10蛋白的分子特征及系统进化，通过荧光定量PCR (qPCR)技术检测该基因在健康中华鳖各组织中的表达分布及其在革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌嗜水气单胞菌胁迫下mRNA的表达规律。结果显示，该蛋白分子质量为10.89 ku，等电点为9.62，存在信号肽和磷酸化位点，单链包含1个N端的loop区、1个α螺旋和3个反向平行β折叠；该基因在健康中华鳖的肝脏和心脏中表达量最高，在血细胞中表达量最低；两种细菌的刺激能促使中华鳖组织器官中cxcl10及炎症相关因子基因tnf-ɑ和il-1β mRNA表达量的显著上调。研究表明，cxcl10可能在中华鳖机体抗菌免疫过程中发挥重要作用。本实验可为中华鳖病害防治提供理论基础。

关键词:中华鳖;CXCL10;mRNA表达;生物信息学

Abstract:

Chemokines are small molecular secreted proteins with chemoattractive properties. They have bactericidal activity and the ability to recruit leukocytes and activate immune cells. CXCL10 is a kind of CXC chemokine with specificity for CXCR3 receptors, which has been shown to mediate the generation of Th1-type immune responses and have antibactericidal effects similar to defensins. Therefore, CXCL10 might be used as an alternative to antibiotics for bacterial diseases in aquaculture industry. In this study, Pelodiscus sinensis was used to study the expression characteristics of cxcl10 and its response to gram-positive bacteria (Staphylococcus aureus) and gram-negative bacteria (Aeromonas hydrophila). The molecular characteristics and systematic evolution of CXCL10 protein in P. sinensis were analyzed by bioinformatics software. The expression and distribution of cxcl10 mRNA in healthy P. sinensis tissues and that after S.aureus or A. hydrophila challenge were detected by quantitative PCR (qPCR). The results showed that the open reading frame of CXCL10 was 303 bp, encoding a protein of 100 amino acids. The molecular weight was 10.89 ku and the estimated isoelectric point was 9.62. P. sinensis cxcl10 had 8 phosphorylation modification sites and a signal peptide. Each single strand of P. sinensis CXCL10 contains one N-terminal loop region, one α helix and three reverse parallel β-folds. Phylogenetic analysis showed a close relationship between P. sinensis CXCL10 with avian and reptile CXCL10s. P. sinensis cxcl10 basal levels were higher in the liver and heart than that in other organs of health turtles. The stimulation of A. hydrophila and S. aureus led to significant upregulation of P. sinensis cxcl10 and inflammation-related factors tnf-ɑ and il-1β in liver, spleen and blood cells. These results suggested that P. sinensis CXCL10 had a similar protein molecular structure with the cationic antimicrobial peptide. P. sinensis cxcl10 was widely expressed in various tissues of P. sinensis, and up-regulated in response to the stimulation of gram-negative bacteria and positive bacteria in immuno-related tissues. In summary, our study found that P. sinensis cxcl10 might be involved in the pro-inflammatory function of antibacterial immune process. These findings are expected to lay a foundation for further research on the antibacterial function of P. sinensis CXCL10 in vitro and in vivo, and also provide a theoretical basis for disease control in P. sinensis breeding industry.

Key words:Pelodiscus sinensis;CXCL10;mRNA expression;bioinformatics