昆虫学报 ›› 2017, Vol. 60 ›› Issue (1): 45-52.doi: 10.16380/j.kcxb.2017.01.006

• 研究论文 • 上一篇    下一篇

家蚕微孢子虫与家蚕ECH1和GAPDH蛋白的相互作用

安欢迎, 王琴, 李治, 党晓群, 周泽扬, 王林玲*   

  1. (重庆师范大学生命科学学院, 重庆 401331)
  • 出版日期:2017-01-20 发布日期:2017-01-20

Interaction between Nosema bombycis and proteins BmECH1 and BmGAPDH in Bombyx mori

AN Huan-Ying, WANG Qin, LI Zhi, DANG Xiao-Qun, ZHOU Ze-Yang, WANG Lin-Ling*   

  1. (School of Life Sciences, Chongqing Normal University, Chongqing 400047, China)
  • Online:2017-01-20 Published:2017-01-20

摘要: 【目的】在分子层面上研究家蚕微孢子虫Nosema bombycis与家蚕Bombyx mori蛋白的相互作用,初步探讨家蚕微孢子虫向家蚕细胞能量中心靠近的原因。【方法】采用Far-western blot分析与家蚕微孢子虫具有相互作用的家蚕中肠蛋白,质谱鉴定筛选出候选蛋白。PCR扩增候选蛋白的基因,连接到pET30a载体并转入大肠杆菌Escherichia coli DH5α感受态细胞培养,测序选取正确的3个重组质粒,转化到大肠杆菌E. coli BL21感受态细胞中诱导表达候选蛋白,亲和层析柱纯化候选蛋白,制备多克隆抗体。用免疫共沉淀和间接免疫荧光技术验证候选蛋白与家蚕微孢子虫的相互作用。【结果】Far-western blot筛选到的anti-SWP9和anti-SWP5抗体与感染家蚕微孢子虫的家蚕中肠总蛋白的PVDF膜孵育,分别在26 kD和34 kD处检测到一条特异条带,说明家蚕微孢子虫与26 kD和34 kD的家蚕中肠蛋白发生了相互作用。对质谱鉴定结果进行蛋白质的分子量、肽段数以及功能的分析,筛选出与家蚕微孢子虫相互作用的候选家蚕蛋白烯酰辅酶A水合酶(ECH1)、甘油醛-3-磷酸脱氢酶(GAPDH)和3-羟酰辅酶A脱氢酶(HCDH)。利用制备的能够特异识别ECH1,GAPDH和HCDH 3种蛋白的多克隆抗体anti-ECH1, anti-GAPDH和anti-HCDH进行免疫共沉淀,证实了家蚕微孢子虫与家蚕中肠蛋白ECH1和GAPDH具有相互作用;间接免疫荧光分析结果进一步说明GAPDH能与家蚕微孢子虫特异性结合。【结论】家蚕微孢子虫可以和家蚕蛋白ECH1和GAPDH特异性结合。由于ECH1是定位于线粒体膜上的脂肪酸β-氧化的关键酶,GAPDH是糖酵解途径的关键酶,推测家蚕微孢子虫可能通过和家蚕ECH1和GAPDH的相互作用,在空间上靠近宿主细胞的线粒体和糖酵解途径,便于摄取宿主细胞脂肪酸β-氧化和糖酵解途径产生的中间产物和ATP,满足家蚕微孢子虫的物质和能量需求。

关键词: 家蚕微孢子虫; 家蚕, 中肠蛋白; 烯酰辅酶A水合酶; 甘油醛-3-磷酸脱氢酶; 蛋白质相互作用

Abstract: 【Aim】 This study aims to study the interaction between Nosema bombycis and proteins in the silkworm, Bombyx mori at the molecular level, and to explore the reason that N.bombycis is close to the energy center in B.mori cells. 【Methods】 Far-western blot analysis was used to search the silkworm midgut proteins that can interact with microsporidia, and the proteins were identified by mass spectrometry. PCR was used to amplify the candidate genes, which were then ligated into pET30a vector and transferred into Escherichia coli DH5α competent cells.The correct recombinant plasmids were selected and transformed into E.coli BL21 competent cells to induce the expression of the candidate proteins.The candidate proteins were purified by affinity chromatography to prepare polyclonal antibodies. The protein interaction was tested and verified by co-precipitation and indirect immunofluorescence techniques. 【Results】 Far-western blot result showed that when anti-SWP9 and anti-SWP5 were incubated with N.bombycis infected silkworm midgut proteins, specific bands of 26 kD and 34 kD appeared, respectively, indicating the interaction between N. bombycis and silkworm midgut proteins (26 kD and 34 kD). Enoyl-CoA hydratase precursor 1 (ECH1), glycerol-3-phosphate dehydrogenase-1 (GAPDH) and 3-hydroxyacyl-CoA dehydrogenase (HCDH), which could interact with N.bombycis, were identified by mass spectrometry analysis on molecular weight, peptide number and function. By using the obtained antibodies anti-ECH1, anti-GAPDH and anti-HCDH, specifically for proteins ECH1, GAPDH and HCDH, respectively, co-immunoprecipitation analysis showed that N.bombycis had interactions with BmECH and BmGAPDH, which could be combined together to precipitate. Indirect immunofluorescence results further showed that BmGAPDH1 could specifically bind to N. bombycis. 【Conclusion】  BmECH1 and BmGAPDH can combine with N. bombycis. ECH1 is the key enzyme of fatty acid beta oxidation, which is localized on the mitochondrial membrane. GAPDH is a key enzyme in the pathway of glycolysis. Through the interaction between N.bombycis and proteins BmECH1 and BmGAPDH, it is speculated that N.bombycis is close to the host cell mitochondria and glycolysis, and the approach lets intermediate products and ATP uptake become more convenient. The intermediate products and ATP can meet the matter and energy demands of N. bombycis.

Key words: Nosema bombycis, Bombyx mori, midgut proteins, enoyl-CoA hydratase precursor 1 (ECH1), glycerol-3-phosphate dehydrogenase-1 (GAPDH), protein interaction