虾肝肠胞虫水通道蛋白基因EHP00_492的克隆与表达特征分析

Cloning and Expression Characteristics Analysis of the Aquaporin Gene EHP00_492 from Enterocytozoon hepatopenaei

【目的】克隆虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)水通道蛋白编码基因EHP00_492,分析其在EHP成熟孢子中的表达特征,为研究EHP水通道蛋白的功能提供理论基础。【方法】以虾肝肠胞虫为材料,克隆获得完整的EHP00_492基因,对其进行序列特征分析,构建pCold-TF-EHP00_492重组表达载体,转化至大肠杆菌中异源表达重组蛋白,制备兔多克隆抗体。利用蛋白免疫印迹实验和间接免疫荧光实验分析EHP00_492在EHP成熟孢子中的表达定位特征。【结果】EHP00_492的编码基因全长729 bp,由242个氨基酸组成,富含亮氨酸,预测分子量为25 kD,无信号肽,含有6个跨膜结构域,具有MIP蛋白家族结构域和2个水通道蛋白保守的NPA/G基序。此外,EHP00_492与其他微孢子虫水通道蛋白序列同源性较高,系统进化树分析结果显示,EHP00_492与毕氏肠胞虫EBI_27080蛋白亲缘关系最近。AlphaFold分析发现,EHP00_492与已鉴定的家蚕微孢子虫水通道蛋白NbAQP和兔脑炎微孢子虫水通道蛋白EcAQP的蛋白质三维结构高度相似,推测EHP00_492是一个潜在的水通道蛋白。蛋白免疫印迹结果显示,EHP00_492蛋白在EHP成熟孢子中有表达,分子量为21 kD。亚细胞定位分析结果显示,EHP00_492蛋白定位于EHP成熟孢子的孢壁上。【结论】初步明确了EHP00_492蛋白的序列特征、结构特征及其与其他微孢子虫水通道蛋白的亲缘关系,以及其在EHP成熟孢子中的表达定位特征,可为进一步研究EHP水通道蛋白的功能提供基础。

【Objective】The aquaporin gene EHP00_492 from Enterocytozoon hepatopenaei(EHP)was cloned and its expression characteristics in EHP mature spores was analyzed,which provided a theoretical basis for studying the function of EHP aquaporin.【Method】The complete EHP00_492 gene was cloned from Ent.hepatopenaei.The sequence characteristics of EHP00_492 was analyzed by bioinformatics methods.Recombinant expression vector pCold-TF-EHP00_492 was constructed and transformed into Escherichia coli to induced fusion protein expression for preparing the rabbit polyclonal antibody of EHP00_492.The expression and subcellular localization characteristics of EHP00_492 in EHP mature spores were analyzed by Western blot and indirect immunofluorescence assay.【Result】EHP00_492 gene is 729 bp in length,encoded 242 amino acids.EHP00_492 is rich in leucine,with a predicted molecular weight of 25 kD.It has no signal peptide,but involving six transmembrane domains,the MIP conserved domain and two aquaporin conserved NPA/G motif.In addition,EHP00_492 has a high homology with other microsporidian aquaporins,and the phylogenetic tree analysis result shows that EHP00_492 is closely related with EBI_27080 protein from Ent.bieneusi.The three-dimensional structure of EHP00_492 is highly similarity with the identified aquaporin NbAQP from Nosema bombycis and EcAQP from Enc.cuniculi by AlphaFold analysis,which is speculated that EHP00_492 is a potential aquaporin.EHP00_492 is a 21 kD-protein in the mature spores of EHP by Western blot analysis.And the subcellular localization analysis showed that EHP00_492 protein was localized at the spore wall of EHP mature spores.【Conclusion】In this study,the sequence and structure characteristics of EHP00_492 protein,as well as its phylogenetic relationship with other microsporidian aquaporins,and the expression and subcellular localization characteristics of EHP00_492 protein in EHP mature spores were preliminary clarified,which provided basis for further study on the function of EHP aquaporins.