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