›› 2014, Vol. 57 ›› Issue (3): 300-307.doi:

• 研究论文 • 上一篇    下一篇


党晓群1,#, 林立鹏2,#, 李春峰2, 潘国庆2, 李田2, 龙梦娴2, 周泽扬1,2,*     

  1. (1. 重庆师范大学动物生物学重点实验室,重庆 401331;
    2. 西南大学,家蚕基因组生物学国家重点实验室,重庆 400716)
  • 出版日期:2014-03-20 发布日期:2014-03-20

Prokaryotic expression and subcellular localization of ADP/ATP carrier protein in microsporidian Nosema bombycis

DANG Xiao-Qun1,#, LIN Li-Peng2,#, LI Chun-Feng2, PAN Guo-Qing2, LI Tian2, LONG Meng-Xian2, ZHOU Ze-Yang1,2,*   

  1. (1. Laboratory of Animal Biology, Chongqing Normal University, Chongqing 401331, China; 2. The State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716, China)
  • Online:2014-03-20 Published:2014-03-20

摘要: 【目的】家蚕微孢子虫Nosema bombycis ADP/ATP转运蛋白可能参与搬运宿主细胞的能量。本研究克隆家蚕微孢子虫ADP/ATP转运蛋白基因,并进行原核表达、抗体制备及间接免疫荧光定位,为控制和防治家蚕微粒子病提供理论基础。【方法】通过同源序列比对鉴定家蚕微孢子虫N. bombycis ADP/ATP转运蛋白序列,采用生物合成的方法将编码3段面向膜内侧肽段的核酸序列拼接合成,在其两端引入BglⅡ和SalⅠ酶切位点,克隆至pUC57载体并测序,再亚克隆至含有二氢叶酸还原酶(dihydrofolate reductase,DHFR)标签的表达载体pQE40中,然后利用BamHⅠ和SalⅠ酶切获得含有DHFR标签的重组序列,并连接至pET30a(+)载体中进行诱导表达。通过SDS-PAGE、镍柱亲和层析和免疫印迹法鉴定表达蛋白,利用间接免疫荧光对ADP/ATP转运蛋白的分布进行检测。【结果】家蚕微孢子虫的ADP/ATP转运蛋白编码序列(GenBank登录号为EOB13854.1)全长1 524 bp,编码蛋白含有507个氨基酸残基,预测分子质量为59 kDa,等电点为9.35。具有12个跨膜结构域和TLC结构域,其中TLC结构域含有4个功能保守位点。与蜜蜂微孢子虫的ADP/ATP转运蛋白比较,氨基酸序列一致性达30%。系统进化分析表明微孢子虫ADP/ATP转运蛋白聚为一类,具有共同的起源。成功构建了NbADP/ATP-△TM-DHFR-pET30a原核表达重组质粒,目的基因获得表达,其融合蛋白分子量约为37 kDa,纯化重组蛋白并制备了多克隆抗体。免疫印迹分析表明,成熟微孢子虫中表达ADP/ATP转运蛋白;间接免疫荧光定位结果显示,家蚕微孢子虫孢子ADP/ATP转运蛋白定位于孢子质膜上。【结论】本研究将为阻断微孢子虫能量来源,达到控制和防治家蚕微粒子病提供新的思路。

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