曼氏迭宫绦虫annexinB8的生物信息学分析和基因克隆

Sequence bioinformatics analysis and gene cloning of annexinB8 from Spirometra mansoni

目的通过生物信息学预测曼氏迭宫绦虫annexinB8(SmannexinB8)的生物学特征,及潜在功能和结构,并且进行基因克隆,为下一步SmannexinB8参与宿主免疫调节研究提供依据。方法通过NCBI的ORF finder工具对SmannexinB8的开放阅读框进行分析.利用ExPASy网站进行蛋白的物理化学参数、信号肽、跨膜螺旋、潜在分子生物学功能的预测,通过NCBI/BLAST。对蛋白保守功能域进行检测。不同物种的annexin序列从NCBI网站获取,并利用Vector NTI suit 8.0和TreeView软件进行分析。利用SWISS-MODEL网站和SPDBV 4.10软件分析SmannexinB8蛋白的三维空间结构。此外。对SmannexinB8基因进行扩增,并克隆到原核表达载体pET-28a(+)。结果 SmannexinB8是一个全长基因,编码347个氨基酸。蛋白由4个典型的annexin重复结构域组成,序列当中没有信号肽,是一个稳定的可溶性蛋白分子。三维空间立体结构分析结果显示SmannexinB8是一个保守的蛋白。SmannexinB8与多房棘球绦虫、口膜壳绦虫、细粒棘球绦虫、华支睾吸虫以及人类的annexin基因的同源性分别是68%,67%。65%,46%和40%。分子进化分析显示SmannexinB8与绦虫属的亲源性最近,而与其他物种,如吸虫、哺乳动物亲源性较远。结论 SmannexinB8可能具有抑制磷脂酶A2的活性,促进细胞融合、参与调节免疫反应和离子通道形成的功能,可能在参与宿主免疫调节中起到关键性的作用。

Objective To provide information and solid materials for further research on annexinB8 of Spirometra mansoni （SmannexinBS） which may participate in mediating host immune response. Methods The open reading frame of SmannexinB8 was found with ORF finder tool in NCBI website. ExPASy website was used to predict the physical and chemical parameters of protein, signal peptide, transmembrane helices and potential molecular and biological functions. The conserved domains of the protein were detected by NCBI/BLAST Home. In order to analyze the homology and phylogenetic tree, sequences of annexins from various species were obtained from NCBI website, and the results were analyzed by Vector NTI suit 8.0 and TreeView software. The three-dimensional structure of SmannexinB8 was predicted by SWISS-MODEL and was analyzed by SPDBV 4.10. Moreover, the gene was amplified and cloned into a prokaryotic expression vector pET-28a （＋）. Results SmannexinB8 was full-length gene and encoded 347 amino acid residues. The protein was composed of four annexin repeats without signal peptide and a stably soluble molecule. It was a conservative annexin in three-dimensional structure. SmannexinB8 was homologous to annexins from Echinococcus multilocularis, Hymenolepis microstoma, Echinococcus granulosus, Clonorchis sinensis and Homo sapiens with 68%, 67%, 65%, 46% and 40% identities, respectively. SmannexinB8 clustered with the annexins from the tapeworm genus, but not with other species like trematode and mammal animals. Conclusions SmannexinB8 might have a multitude of functions： inhibition of phospholipase A2, promoting fusion, mediating immune response and formation of ion channel. SmannexinB8 might play a critical role in the host immune re- sponse.