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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 explore the biological characteristics and functions of Clonorchis sinensis growth factor receptor binding protein 2 (CsGrb2). Methods Full-length cDNA of CsGrb2 was obtained from the cDNA library of Clonorchis sinensis, and NCBI was used to search for the gene sequence with high similarity. The MEGA5 program was used to analyze the gene sequence of CsGrb2 from GenBank with high similarity The recombinant protein CsGrb2 was expressed in E.coli. The recombinant fusion protein CsGrb2 was expressed in E.coli. The recombinant fusion protein CsGrb2 was used to immunize mice to produce polyclonal antibody. The natural CsGrb2 was identified by Western blot in the extract of CsCl. The distribution of CsGrb2 in adults and metacercariae of Clonorchis sinensis was detected by histochemical method. The expression level of CsGrb2 gene in different developmental stages was determined by qRT-PCR. Results The CsGrb2 gene encoded a polypeptide containing SH3-SH2-SH3 structure, which had a high similarity with Grb2 in human and various animals and had a closer evolutionary relationship with other flukes. The recombinant CsGrb2 was highly expressed in prokaryotic system and purified The results of Western blot showed that the natural CsGrb2 and recombinant CsGrb2 had the same molecular mass of 34 ku, while CsGrb2 had higher expression level in metacercaria than adult. Immunohistochemistry showed CsGrb2 mainly distributed in the mouth of adult, abdominal suction, interstitial tissue, spermatozoa and metacercaria in most of the organs. Conclusion CsGrb2 belongs to the conserved protein, suggesting that it plays a role in the organogenesis, energy metabolism and sperm mitosis of Clonorchis sinensis.