| 李晨曦 1，吴慧欣 1,2，吴玉娇 1，陈 洁 1，孟宪志 1，潘国庆 1，龙梦娴 1.虾肝肠胞虫水通道蛋白基因 EHP00_492 的克隆与表达特征分析[J].广东农业科学,2023,50(8):154-162 |
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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 |
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| DOI：10.16768/j.issn.1004-874X.2023.08.016 |
| 中文关键词: 虾肝肠胞虫 水通道蛋白 基因克隆 序列特征 表达 亚细胞定位 |
| 英文关键词: Enterocytozoon hepatopenaei aquaporin gene cloning sequence characteristics expression subcellular localization |
| 基金项目:重庆市自然科学基金创新群体项目（cstc2021jcyj-cxttX0005） |
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| 中文摘要: |
| 【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。 |
| 英文摘要: |
| 【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. |
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