华支睾吸虫生长因子受体结合蛋白2的生物学特性和功能研究

A study on the characteristics and function of Clonorchis sinensis growth factor receptor-bound protein 2

目的探索华支睾吸虫生长因子受体结合蛋白2(Clonorchis sinensis growth factor receptor bound protein 2,CsGrb2)的生物学特性和功能。方法从华支睾吸虫cDNA文库获得CsGrb2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程序对来自于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CsGrb2,用表达的融合蛋白CsGrb2免疫小鼠,产生多克隆抗体,采用Western blot鉴定华支睾吸虫提取物中的天然CsGrb2,采用免疫组化法检测CsGrb2在华支睾吸虫成虫及囊蚴的分布;采用qRT-PCR测定CsGrb2基因在不同发育阶段的表达水平。结果 CsGrb2基因编码一个含有SH3-SH2-SH3结构的多肽,与人及各种动物的Grb2有着很高的相似度,与其他吸虫有着更近的进化关系;重组CsGrb2在原核系统中大量表达并纯化,Western blot显示天然CsGrb2与重组CsGrb2分子质量相同,均为34ku;CsGrb2在囊蚴中的表达量高于成虫。免疫组化显示CsGrb2主要分布于成虫的口、腹吸盘,间质组织,受精囊中的精子及囊蚴的大多数器官。结论CsGrb2属于保守蛋白,推测在华支睾吸虫的器官生成,能量代谢和精子的有丝分裂方面发挥作用。

Objective To determine the molecular characteristics of growth factor receptor-bound protein 2（Grb2）from Clonorchis sinensis（CsGrb2）in contrast to Grb2 from other animals and its possible functions in the development and organogenesis of C.sinensis. Methods The full-length cDNA of CsGrb2 was retrieved from the C.sinensis transcriptome and was used to search for similar sequences in the NCBI database,and the domains and functions of CsGrb2 were predicted.MEGA5 was used to produce a phylogenetic tree for genes that are highly similar to CsGrb2 from GenBank.Recombinant CsGrb2 protein was expressed in a prokaryotic system and was used to immunize mice to produce polyclonal antibodies.Western blotting was used to identify native CsGrb2 from an extract of C.sinensis.Immunohistological staining and qRT-PCR were used to detect the distribution of CsGrb2 in metacercariae and adult C.sinensis and its level of expression in different stages of development. Results A CsGrb2 cDNA clone retrieved from the C.sinensis transcriptome encoded a polypeptide with a SH3-SH2-SH3 structure and was grouped with homologs from trematodes.Recombinant CsGrb2 was produced in bacteria and purified to homogeneity.Native CsGrb2 with an estimated molecular weight of 34 ku was identified from an extract of adult C.sinensis.CsGrb2 transcripts were more abundant in metacercariae than in adults.Immunohistochemical staining indicated that CsGrb2 was localized to the suckers,mesenchymal tissues,sperm in the seminal receptacle,and ovary in adults and that CsGrb2 was abundantly expressed in most organs of the metacercariae. Conclusion Grb2 protein found in C.sinensis was conserved among different animals,suggesting that Grb2 plays a role in the organogenesis,energy metabolism,and mitotic spermatogenesis of C.sinensis.These findings fromC.sinensis provide greater understanding of the varied functions of Grb2 in lower animals such as platyhelminths.