昆虫学报 ›› 2021, Vol. 64 ›› Issue (9): 1070-1079.doi: 10.16380/j.kcxb.2021.09.007

• 研究论文 • 上一篇    下一篇

东方蜜蜂微孢子虫一新型孢壁蛋白的基因克隆、 表达及亚细胞定位

熊亮#, 敖塘堰#, 张真, 马振刚*, 周泽扬   

  1.  (重庆师范大学, 重庆市动物生物学重点实验室, 重庆市媒介昆虫重点实验室, 重庆 401331)
  • 出版日期:2021-09-20 发布日期:2021-09-03

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

摘要:

【目的】微孢子虫(Microsporidia)孢壁在孢子构成及孢子侵染宿主过程中扮演重要的角色。本研究旨在鉴定获得的东方蜜蜂微孢子虫Nosema ceranae新型孢壁蛋白,并进行基因克隆和原核表达,明确其亚细胞定位。【方法】通过在线软件对东方蜜蜂微孢子虫新型孢壁蛋白AAJ76_1400036761序列进行生物信息学分析。利用PCR法获取目的片段并将其克隆至原核表达载体pCold II中,利用IPTG诱导表达重组蛋白并通过镍柱亲和层析法纯化目的蛋白。以获得的重组蛋白为抗原免疫小鼠制备多克隆抗体,通过间接免疫荧光技术和免疫胶体金定位技术对该蛋白进行亚细胞定位分析;利用蛋白质免疫印迹法检测该蛋白与东方蜜蜂微孢子虫几丁质壳的互作。【结果】在MicrosporidiaDB数据库中获得AAJ76_1400036761基因序列,基因全长681 bp,编码226个氨基酸;预测等电点为6.84,分子量为26.19 kD。SDS-PAGE电泳和Western blot结果表明AAJ76_1400036761重组蛋白能够在大肠杆菌Eescherichia coli Rosetta中高量表达。Western blot结果表明,制备的多克隆抗体能够特异地识别东方蜜蜂微孢子虫总蛋白中的AAJ76_1400036761,说明其在成熟东方蜜蜂微孢子虫中有表达。亚细胞定位结果显示,AAJ76_1400036761定位于东方蜜蜂微孢子虫孢壁上。重组蛋白AAJ76_1400036761能够与蜜蜂微孢子虫的几丁质壳结合。【结论】AAJ76_1400036761蛋白在东方蜜蜂微孢子虫成熟孢子中有表达;该蛋白定位于东方蜜蜂微孢子虫孢壁上,为东方蜜蜂微孢子虫新的孢壁蛋白。本研究为深入研究该蛋白的生物学功能奠定了基础。

关键词: 东方蜜蜂微孢子虫, 孢壁蛋白, 原核表达, 多克隆抗体, 亚细胞定位

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