Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella
Molecular characterization and analysis of a novel calcium-dependent protein kinase from Eimeria tenella
复制标题
柔嫩艾美耳球虫新型钙依赖性蛋白激酶的分子表征和分析
DOI：
10.1017/s0031182012002107
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发表时间：
2013-05-01
期刊：
影响因子：
2.4
通讯作者：
Huang, B.
中科院分区：
文献类型：
--
作者：
Han, H. Y.;Zhu, S. H.;Huang, B.
摘要钙依赖蛋白激酶(CDPKs)是仅存在于植物、绿藻、纤毛虫和顶复合体寄生虫中的一种独特的酶。本研究利用基于表达序列标签(EST)的快速扩增技术(RACE)克隆了柔嫩艾美耳球虫CDPK新基因EtCDPK3。EtCDPK3的全长为1637个核苷酸，编码433个氨基酸，推导出的EtCDPK3蛋白具有与其他CDPKs相同的典型特征结构域，包括一个保守的氨基末端的激酶结构域和一个具有4个EF手性钙结合基序的羧基末端的类钙调素结构。采用实时荧光定量聚合酶链式反应技术检测EtCDPK3基因在不同发育阶段的表达谱。EtCDPK3基因的信使RNA水平在子孢子中高于其他时期(未孢子化卵囊、孢子化卵囊和裂殖子)。Western印迹分析表明，重组EtCDPK3兔抗血清可识别寄生虫天然的49 kDa蛋白条带。间接免疫荧光抗体标记显示，EtCDPK3在第一次分裂过程中定位分散，特异性强。早期感染DF-1细胞后，EtCDPK3定位于子孢子顶端，并高表达。使用特定抗体抑制EtCDPK3功能降低了柔嫩艾美耳球虫入侵宿主细胞的能力。这些结果提示，EtCDPK3可能参与了柔嫩艾美耳球虫胞内期的侵袭和存活。由于该激酶家族不存在于宿主中，因此它代表了一个有效的靶点，可用于治疗艾美耳球虫。
SUMMARY The calcium-dependent protein kinases (CDPKs) are unique enzymes found only in plants, green algae, ciliates and apicomplexan parasites. In this study, a novel CDPK gene of Eimeria tenella, designed EtCDPK3, was cloned using rapid amplification of cDNA ends (RACE) based on the expressed sequence tag (EST). The entire cDNA of EtCDPK3 contained 1637 nucleotides encoding 433 amino acids and the deduced EtCDPK3 protein had canonical characteristic domains identified in other CDPKs, including a well-conserved amino-terminal kinase domain and a carboxy-terminal calmodulin-like structure with 4 EF-hand motifs for calcium binding. The expression profiles of the EtCDPK3 gene in different development stages were investigated by real-time quantitative PCR. Messenger RNA levels from the EtCDPK3 gene were higher in sporozoites than in other stages (unsporulated oocysts, sporulated oocysts and merozoites). Western blot analysis showed that rabbit antiserum against recombinant EtCDPK3 could recognize a native 49 kDa protein band of parasite. Indirect immunofluorescent antibody labelling revealed dispersed localization of EtCDPK3 during the first schizogony and intense specific staining. EtCDPK3 was located at the apical end of the sporozoites after early infection of DF-1 cells and the protein was highly expressed. Inhibition of EtCDPK3 function using specific antibodies reduced the ability of E. tenella to invade host cells. These results suggested that EtCDPK3 may be involved in invasion and survival of the parasite intracellular stages of E. tenella. Because this kinase family is absent from hosts, it represents a valid target that could be exploited for chemotherapy against Eimeria spp.