Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella.
Molecular Characterization and Functional Analysis of a Novel Calcium-Dependent Protein Kinase 4 from Eimeria tenella.
复制标题
柔嫩艾美球虫新型钙依赖性蛋白激酶 4 的分子表征和功能分析
DOI：
10.1371/journal.pone.0168132
复制
发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Han H
中科院分区：
文献类型：
--
作者：
Wang Z;Huang B;Dong H;Zhao Q;Zhu S;Xia W;Xu S;Xie Y;Cui X;Tang M;Men Q;Yang Z;Li C;Zhu X;Han H
柔嫩艾美耳球虫（Eimeria tenella）是一种专性细胞内寄生虫，活跃地侵入鸡盲肠上皮细胞。细胞侵袭的基础还不完全清楚，但已经发现了一些宿主细胞侵袭的关键分子。本文研究了大肠杆菌中钙依赖性蛋白激酶4（EtCDPK 4）的特性。tenella入侵宿主细胞。从E. tenella使用cDNA末端的快速扩增。EtCDPK 4的开放阅读框为1803 bp，编码600个氨基酸。采用实时荧光定量PCR和Western blotting方法研究了EtCDPK 4在大肠杆菌4个发育阶段转录和翻译的差异。tenella。EtCDPK 4在子孢子中以较高水平表达，但在第二代裂殖子中翻译较高。体外侵袭抑制实验探讨EtCDPK 4是否参与E.柔嫩孢子针对重组EtCDPK 4（rEtCDPK 4）的多克隆抗体抑制寄生虫的侵袭，使其降低约52%。间接免疫荧光法研究了E. DF-1细胞的tenella子孢子体外侵袭。结果表明，EtCDPK 4可能在子孢子的侵入和发育过程中起重要作用。为了根据EtCDPK 4的结构特点分析其功能结构域，研究rEtCDPK 4的激酶活性，建立了体外磷酸化体系。我们验证了rEtCDPK 4是一种完全依赖于Ca 2+的蛋白激酶。通过体外激酶活性筛选rEtCDPK 4活性的特异性抑制剂。应用某些特异性抑制剂检测E. tenella子孢子，以证实抑制剂在体外起作用。W-7、H-7、H-89和肉豆蔻酰肽抑制E.柔嫩孢子实验结果表明，EtCDPK 4可能参与了E.柔嫩体侵入鸡盲肠上皮细胞。
Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.
登录
查看更多内容
大豆钙依赖性蛋白激酶的全基因组鉴定及其对昆虫食草和干旱胁迫的转录反应分析
影响因子：
4.6
作者：
Hettenhausen C;Sun G;He Y;Zhuang H;Sun T;Qi J;Wu J
通讯作者：
Wu J
影响因子：
4
作者：
Entzeroth, R;Mattig, FR;Werner-Meier, R
通讯作者：
Werner-Meier, R
影响因子：
3.4
作者：
Belli, SL;Walker, RA;Flowers, SA
通讯作者：
Flowers, SA
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
影响因子：
2.4
作者：
Han, H. Y.;Zhu, S. H.;Huang, B.
通讯作者：
Huang, B.
影响因子：
7.4
作者：
Dammann, C;Ichida, A;Harper, JF
通讯作者：
Harper, JF