华支睾吸虫钙调蛋白基因的功能预测及其在华支睾吸虫成虫活动和囊蚴脱囊中的作用探讨

Bioinformatic analysis of the calmodulin of Clonorchis sinensis and the possible role of calmodulin in worm activity and excystation of Clonorchis sinensis

目的采用生物信息学方法分析预测华支睾吸虫钙调蛋白(CsCaM)基因的结构和功能,通过体内实验观察其对华支睾吸虫成虫活动能力和囊蚴脱囊的影响。方法利用生物信息学方法对华支睾吸虫钙调蛋白基因及其对应蛋白质的理化性质和空间构象进行预测,并通过对其他CaM序列进行同源性比对,分析该基因的进化规律。用CaM抑制剂三氟拉嗪(TFP)与华支睾吸虫成虫、囊蚴分别孵育,观察成虫活动能力以及囊蚴脱囊情况。结果CsCaM基因编码区在第93-543 bp,编码149个氨基酸。编码蛋白的等电点为4.14,理论相对分子质量为16.8×103。拓扑结构分析该蛋白无信号肽和跨膜区。InterProScan预测CsCaM含2个超家族,4个EF手性结构域(1个超家族含有2个EF结构域),分别位于aa21-aa33,aa57-aa69,aa94-aa106和aa130-aa142。CsCaM与其他物种CaM的同源性均在90%以上。TFP与华支睾吸虫成虫和囊蚴共孵育后,实验组成虫活动能力和囊蚴脱囊率均较对照组有所下降。结论CsCaM具有钙离子信号蛋白的特性,参与华支睾吸虫成虫活动和囊蚴脱囊,是华支睾吸虫生长发育过程中的重要蛋白质。

Objective To predict the structural and functional characteristics of calmodulin of Clonorchis sinensis(CsCaM)and determine whether CsCaM may participate in movement of adult worms and excystation of metacercariae.Methods The physicochemical properties,conserved domains,signal peptides,hydrophilicity and hydrophobicity,homology,and amino acid sequence of CsCaM were predicted using bioinformatic software such as InterProScan,PredictProtein,SignalP,Vector NTI suite 8.0,and MEGA 5.0.Adult flukes were freshly harvested and then incubated in the different concentrations of the CaM antagonist trifluoperazine(TFP).Worm activity was observed.C.sinensis metacercariae were collected and then incubated with an excystation solution containing TFP.The number of excysted metacercariae was determined microscopically.Results CsCaM contained an ORF coding a putative protein of 149 amino acids.CsCaM has a predicted molecular mass of 16.8×103 and an isoelectric point(PI)of 4.14.No signal peptides or trans-membrane domains were found.The protein’s secondary structure consisted ofα-helices(59.06%)and loops(40.94%).Four conserved EF-hand domains with Ca2+-binding were located at amino acids 21-33,57-69,94-106,and 130-142 in CsCaM.Worm activity was lower with exposure to a high concentration of TFP than with exposure to a low concentration.Moreover,metacercariae excysted at a lower rate with exposure to a high concentration of TFP than with exposure to a low concentration.Conclusion Bioinformatic analysis indicated that CsCaM has the properties of a calcium ion signaling protein and that TFP can inhibit worm activity as well as excystation,indicating that a CaM-dependent process is involved in worm activity and excystation.