华支睾吸虫parkin蛋白的分子生物学特征研究

Research to characterization the parkin protein of Clonorchis sinensis

目的了解华支睾吸虫Parkin蛋白(Clonorchis sinensis Parkin)的生物学特性。方法从华支睾吸虫cDNA文库获得Parkin的cDNA全长,NCBI搜索与之相似度高的基因序列,预测其所含结构域和功能;应用MEGA5程序对来自GenBank中与之有较高相似度的基因序列进行分析,构建基因进化树;原核表达重组融合蛋白CsParkin,纯化后免疫Blab/c小鼠,获得多克隆抗体,采用Western blot鉴定天然CsParkin;采用qRT-PCR测定CsParkin基因在不同发育阶段的表达水平。结果 CsParkin蛋白含有5个结构域,分别是Ubl、RING0、RING1、IBR和RING2,具有典型的Parkin蛋白结构;该蛋白与人及各种动物的Parkin有很高的相似度,与其他吸虫有更近的进化关系;Western blot显示原核表达的重组CsParkin,与天然CsParkin分子质量相同,均为45.7ku;CsParkin在囊蚴中的表达量高于成虫;免疫组化显示CsParkin主要分布于成虫受精囊中的精子及口吸盘和睾丸,在囊蚴中广泛分布。结论肝吸虫CsParkin属蛋白于保守蛋白,具有典型的泛素连接酶3的功能结构,推测其在蛋白的泛素化中起到作用,可能在华支睾吸虫的生长过程中发挥作用。

Objective To study the molecular characteristics of the parkin protein fromClonorchis sinensis（C.sinensis parkin）. Methods The full-length cDNA of C.sinensis parkin was retrieved from the C.sinensis transcriptome.The NCBI database was searched for similar sequences,and the domains and functions of the parkin protein were predicted.MEGA5 was used to construct a phylogenetic tree for similar genes coding for C.sinensis parkin in GenBank.Recombinant C.sinensis parkin protein was expressed in a prokaryotic system and purified.The recombinant protein was then used to immunize mice to obtain polyclonal antibody.Native C.sinensis parkin was identified with Western blotting.Immunohistological staining and qRT-PCR were used to detect the distribution of C.sinensis parkin in metacercariae and adults and to determine its level of expression in different stages of development of C.sinensis. Results C.sinensis parkin consists of five domains including Ubl,RING0,RING1,IBR,and RING2.C.sinensis parkin has a structure typical of other similar parkin proteins and is related to homologs in other trematodes.Native parkin from adult C.sinensis was found to have an estimated molecular weight of 45.7ku according to Western blotting.Parkin transcripts were more abundant in metacercariae than in adults.Immunohistochemical staining indicated that parkin was located in sperm in the seminal receptacle and in the oral sucker and testes in adults,and it was widely distributed in metacercariae. Conclusion Parkin was conserved among trematodes like C.sinensis and it has functions and a structure typical of ubiquitin ligase E3,suggesting that parkin plays a role in ubiquitination of protein in C.sinensis and the development of that trematode.