A New Spore Wall Protein 9 Gene Cloned from Nosema pernyi (Microsporidia) Isolated from Chinese Oak Silkworm, Antheraea pernyi

Abstract

Aims: China has plenty of oak trees that form the cradle of tussah (Antheraea pernyi) industry. Pebrine is a serious disease along with tussah rearing and is difficult to solve. This pathogen named Nosema pernyi, which can infect the Chinese oak silkworm. The spores of N. pernyi have thick spore wall constructed by exospore and endospore. Spore wall proteins contact with host cells are related to microsporidia infection. Methodology: In this study, we used the percoll gradient centrifugation method to purify spores of N. pernyi. Electron microscopy was used to detect the spore wall structure. Recombinant prokaryotic expression vector was constructed and induced in Escherichia coli. SDS-PAGE and Western blot (WB) was performed to detect the protein expression. Results: A gene was cloned including an open reading frame (ORF) of 954 bp coding for a theoretical 317 amino acids protein. BLASTp showed a high amino acid sequence homology with spore wall protein in other microsporidia species. We named this gene NpSWP9, and a prokaryotic expression vector was constructed. Recombinant plasmid of NpSWP9-E1 Vector was transferred into Transetta (DE3). According to SDS-PAGE result, the molecular weight of the target protein was 38 kDa under the condition of IPTG (Isopropyl β-D-Thiogalactoside) for 5 h. According to the WB result, about 40 kDa band was detected by anti-HIS tag antibody. Conclusion: A new spore wall protein gene was identified in N. pernyi from A. pernyi. This research provided a good basis for further studies on cellular localization and immunodetection of NpSWP9 in N. pernyi.