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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 understand the biological characteristics of Clonorchis sinensis Parkin. Methods The full-length cDNA of Parkin was obtained from the cDNA library of Clonorchis sinensis, and the NCBI was used to search for the gene sequence with high similarity. The MEGA5 program was used to analyze the nucleotide sequence of Parkin gene from GenBank with high similarity The recombinant protein CsParkin was expressed in prokaryotic cells. After purification, Blab / c mice were immunized to obtain polyclonal antibodies. The native CsParkin was identified by Western blot. The CsParkin gene was detected by qRT-PCR at different developmental stages The expression level. Results The CsParkin protein contained five domains, namely, Ubl, RING0, RING1, IBR and RING2, which had the typical Parkin protein structure. The protein had high similarity with Parkin in humans and animals, The results of Western blot showed that the expressed recombinant CsParkin was 45.7ku, which was the same as that of natural CsParkin. The expression level of CsParkin in metacercaria was higher than that in adults. Immunohistochemistry showed that CsParkin was mainly distributed in the adult seminal vesicles Sperm and mouth sucker and testis, widely distributed in metacercariae. Conclusion CsParkin is a conserved protein with the functional structure of typical ubiquitin ligase 3. It is presumed that CsParkin plays a role in ubiquitination of proteins and may play a role in the growth of Clonorchis sinensis.