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| 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 |
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| Abstract: |
| 【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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