Acta Entomologica Sinica ›› 2021, Vol. 64 ›› Issue (9): 1070-1079.doi: 10.16380/j.kcxb.2021.09.007

• RESEARCH PAPERS • Previous Articles     Next Articles

Gene cloning, expression and subcellular localization of a novel spore wall protein of Nosema ceranae (Microsporidia)

XIONG Liang#, AO Tang-Yan#, ZHANG Zhen, MA Zhen-Gang*, ZHOU Ze-Yang   

  1.  (Chongqing Key Laboratory of Animal Biology, Chongqing Key Laboratory of Vector Insect, Chongqing Normal University, Chongqing 401331, China)
  • Online:2021-09-20 Published:2021-09-03

Abstract: 【Aim】 The spore wall of Microsporidia plays an important role in the process of spore formation and host infection. The objective of this study is to identify a novel spore wall protein in Nosema ceranae, and to conduct the gene cloning and prokaryotic expression in order to clarify the subcellular localization of this protein. 【Methods】 Bioinformatics analysis of a novel spore wall protein AAJ76_1400036761 in N. ceranae was carried out using online software. The target DNA fragment was obtained by PCR and then cloned into the prokaryotic expression vector pCold II. The recombinant protein was induced by IPTG and purified by Ni2+ affinity chromatography. Polyclonal antibody was prepared by immunizing mice with the recombinant protein as antigen. The subcellular localization of the protein was analyzed by indirect immunofluorescence and immuno-colloidal gold labelling. Western blotting was used to detect the interaction between the protein and the chitin spore coat of N. ceranae. 【Results】 The gene sequence of AAJ76_1400036761 was obtained from Microsporidia DB. It is 681 bp in length encoding 226 amino acids with the predicted isoelectric point of 6.84 and molecular weight of 26.19 kD. The results of SDS-PAGE and Western blotting showed that the recombinant protein could be highly expressed in Escherichia coli Rosetta cells. Western blotting result revealed that the prepared polyclonal antibody could specifically recognize AAJ76_140003676 in the total proteins of N. ceranae, suggesting that this protein is expressed in mature spores of N. ceranae. The subcellular localization analysis showed that AAJ76_140003676 is located at the spore wall of N. ceranae. The recombinant protein AAJ76_1400036761 could bind with the chitin spore coat of N. ceranae. 【Conclusion】 AAJ76_140003676 is expressed in mature spores of N. ceranae. It is a novel spore wall protein of N. ceranae located at the spore wall. This study lays a foundation for further studying the biological functions of this protein.

Key words:  Nosema ceranae, spore wall protein, prokaryotic expression, polyclonal antibody, subcellular localization