关键词: 家蚕微孢子虫, ADP/ATP转运蛋白, 原核表达, 免疫印迹, 亚细胞定位

Abstract: 【Aim】 The ADP/ATP carrier protein in microsporidian Nosema bombycis may be involved in carrying the energy from host cells to itself. In order to provide the theoretical basis for preventing and controlling the pebrine disease, the gene of N. bombycis ADP/ATP carrier protein was cloned and expressed in prokaryocytes. The antibody was prepared and indirect immunofluorescence assay was conducted. 【Methods】 The nucleotide sequences encoding three segmental peptides inside the membrane were synthesized with BglⅡ and SalⅠ sites at both ends (NbADP/ATP-△TM) and cloned into pUC57 vector for sequencing, and the target sequence was further subcloned into pQE40 vector with dihydrofolate reductase （DHFR） tag. The NbADP/ATP-△TM-DHFR was cut by BamHⅠ and SalⅠ and linked to pET30 vector for prokaryotic expression. The expression product was identified by SDS-PAGE, Ni-NTA affinity chromatography and immunoblotting. Indirect immunofluorescence assay was performed to survey the distribution of NbADP/ATP. 【Results】 The coding sequence of nbadp/atp (GenBank accession no. EOB13854.1) is 1 524 bp in length, encoding a 507-amino acid residue peptide with a calculated molecular weight of 59 kDa and a theoretical pI of 9.35. The ADP/ATP carrier protein of N. bombycis contains twelve transmembrane domains and the conserved TLC domain which has four conserved functional sites. Sequences blast analysis showed that the ADP/ATP carrier protein of N. bombycis shares 30% amino acid sequence identity with Nosema ceranae ADP/ATP carrier protein 2. Phylogenetic analysis placed the N. bombycis protein in the same subgroup as the ADP/ATP carrier. The recombinant plasmid NbADP/ATP-△TM-DHFR-pET30a was successfully constructed. SDS-PAGE analysis showed that the 37 kDa fusion protein was highly expressed and purified. Anti-NbADP/ATP serum was produced in mice with the purified protein. Immunoblotting result showed that NbADP/ATP was expressed in mature spores. Indirect immunofluorescence localization results revealed that ADP/ATP transporter protein is located in the plasma membrane. 【Conclusion】 This study provides a new clue for blocking energy source of N. bombycis to control and prevent pebrine disease of the silkworm.

Key words: Nosema bombycis, ADP/ATP carrier protein, prokaryotic expression, Western blotting, subcellular localization