论文部分内容阅读
目的获得原核表达的华支睾吸虫Rab2蛋白,并探索其生物学特性。方法从华支睾吸虫cDNA文库获得CsRab2的全长cDNA,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Vector NTI程序对来自于Gen Bank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;Swiss-model预测其三维立体结构,原核表达重组融合蛋白CsRab2,采用Western blot鉴定重组蛋白CsRab2,采用q RT-PCR测定CsRab2基因在不同发育阶段的表达水平。结果 CsRab2全长基因的ORF包含588个碱基,编码195个氨基酸。Prot Param预测该编码蛋白的理论分子量和等电点分别是22.29×103 Mr和5.87。不含有信号肽,也未发现线粒体等亚细胞定位序列,三维预测结果显示,Rab2同Rab家族的其他蛋白类似:由保守的G结构域和长度、序列上高度可变的N端和C端形成。将纯化后的蛋白进行SDS-PAGE电泳,考马斯亮蓝染色观察该目的蛋白分子量约28×103 Mr。经过亲和层析方法纯化获得的rCsRab2,可被His tag小鼠单克隆抗体识别。CsRab2在华支睾吸虫各生活史阶段均有表达,且在囊蚴和尾蚴阶段表达较高。结论 CsRab2属于保守蛋白,其生物信息学分析符合Rab家族蛋白的共有特性,在囊蚴和尾蚴阶段表达较高,但CsRab2在细胞内确切的定位信息和功能仍有待进一步研究。
Objective To obtain the prokaryotic expression of Rab2 protein of Clonorchis sinensis and to explore its biological characteristics. Methods Full-length cDNA of CsRab2 was obtained from the cDNA library of Clonorchis sinensis, NCBI was used to search for the gene sequence with high similarity and to predict the domain and function of CsRab2. The gene sequence was analyzed to construct the phylogenetic tree. The three-dimensional structure was predicted by Swiss-model. The recombinant fusion protein CsRab2 was expressed in prokaryotic cells and the recombinant protein CsRab2 was identified by Western blot. The qRT-PCR was used to detect the expression of CsRab2 gene at different developmental stages The expression level. Results The ORF of CsRab2 full-length gene contained 588 bases and encoded 195 amino acids. Prot Param predicted the theoretical molecular weight and isoelectric point of the encoded protein were 22.29 × 103 Mr and 5.87. No signal peptide was found and no subcellular localization sequence such as mitochondria was found. Three-dimensional prediction showed that Rab2 is similar to other Rab family proteins: the conserved G domain and its length, highly variable N-terminal and C-terminal . The purified protein was subjected to SDS-PAGE electrophoresis, Coomassie blue staining of the target protein molecular weight of about 28 × 103 Mr. The rCsRab2 purified by affinity chromatography can be recognized by His tag mouse monoclonal antibody. CsRab2 was expressed in all life stages of Clonorchis sinensis and was highly expressed in metacercariae and cercariae. Conclusion CsRab2 is a conserved protein. Its bioinformatics analysis accords with the common features of Rab family proteins and has high expression in metacercarial and cercarial stages. However, the exact location and function of CsRab2 remains to be further studied.