Molecular Mechanism Of Locust Microsporidia Infection Host

In the paper, our research want to know the molecular mechanism that how Nosema locustae escaped the immune reaction of Locusta migratoria manilensis Meyen and infect it. we use spore wall protein of Nosema locustae as a target to analyze the pathway that Nosema locustae infected the host L. migratoria manilensis Meyen.1. Using Immobilized pH Gradient two-dimensional electrophoresis technology and MALDI-TOF-MS get a new spore wall protein of Nosema locustae, named AlocSWP2. Its molecular weight is 23 kDa, containing a N glycosylation site、a heparin-binding motifs and four conserved cysteine sites. its signal peptide contains nineteen amino acids.2. Getting the AlocSWP2 gene of Nosema locustae, then we use AloSWP2 gene to construct the prokaryotic expression vector pGEX4T-1-AlocSWP2. 0.5mM IPTG is then used to induce GST-AlocSWP2 expression in E.coli BL21(DE3). After ultrasonication the expressed E.coli BL21(DE3), detected the supernatant and precipitate using SDS-PAGE. All of them contain the target protein GST-AlocSWP2, but GST-AlocSWP2 content in the supernatant is less than the precipitation. The protein In the supernatant is active,while in the precipitation take shape in inclusion body without activity. So we use the supernatant for the purification of GST-AlocSWP2. AlocSWP2 protein was purified by GST Sepharose 4B kit,gaining more pure target protein(containing a small amount of impurity protein,does not affect the subsequent experiment). Then, we use the purified Protein AlocSWP2 to inject New Zealand white rabbit to get the antiserum. Then evaluating its titer by Western blotting, the result show that the antiserum can be used for Immune colloidal gold localization experiment. At the same time, the Immune colloidal gold localization experiment demonstrated that AlocSWP2 protein is indeed located on the spore wall of Nosema Locustae.3. Using RNAi interference 、challenge assay and GST/His-pull down experiment to explore the function of AlocSWP2. The RNAi result show that non- injected dsRNA Locusta migratoria manilensis is 6.6 times higher than the AlocSWP2 expression amount of the injected dsRNA Locusta migratoria manilensis,showing that succeed in RNAi interference. In the challenge assay experiment, Locusta migratoria manilensis show some symptoms: compared with healthy Locusta migratoria manilensis, infected N. locustae of L. migratoria manilensis are difficult to molt, sometimes resulting in death due to molt. Its move slowly, gradually the body turned into a dark brown, until the whole body become red. In the anatomy experiment, observed the midgut of infected N. locustae of L. migratoria manilensis become ulcerate, its fat body will be exhausted. To further confirm that fat body was the main infection site of N. Locustae in Locusta migratoria manilensis. At the same time the challenge assay experiment show that RNAi interference can delay the death of Locusta migratoria manilensis. So, AlocSWP2 protein plays a certain role in the infected host, associated with immune reaction.4. Using purified AlocSWP2 protein as a bait to catch the interactive protein from the host by GST/His-pull down experiment. The result is not obtained the anticipated objective protein, but it provides a good reference for the further study the mechanism that N. Locustae infected the host L. migratoria manilensis Meyen.