Locusts Micro Sporozoan Spore Wall Protein Identification And Function Analysis

Spore wall proteins are a kind of proteins which exist on the most outer layer ofmicrosporidia. Recent studies suggest that their surface proteins play important roles inhost tissue infection and invasion process. In this study, a combination of certainextract methods and two-dimensional electrophoresis techniques were employed by,using mass spectrometry to identify multiple possible spore wall proteins. Andfortunately, the first spore wall protein from Antonospora Locustae was found throughbioinformatics analysis means, named ALSWP1. Then ALSWP1was studied further:including sequence analysis, gene cloning, prokaryotic expression,immunofluorescence(IFA)and immunoelectron microscopy (IEM) analysis wereemployed on protein localization, then the level of RNA expression profiles wereanalyzed. Finally, function analysis of the spore wall protein ALSWP1was conductedthrough GST-pull down and its three-dimensional model. In this dissertation, the mainresults are as follows:Protein extraction was performed on two methods, SDS-boiling and Brossonmethod, respectively. The two extract method was analyzed by a2-DE proteomicmethod, and obtained111protein spots. Wide pH gradient gels (pH3–10) revealedabout100different spots for more than70%of these being composing of the30-50kDa/pI5-8range. However, only SDS-boiling extract were used on the MALDItarget, as well as to data acquisition and interpretation. For some spots, theidentification was confirmed or obtained by LC-MS/MS analysis.As a result,5proteins were identified, and the22.4kDa putative spore wall protein were selected tostudy after analyzing these proteins.Researches were carried on gene cloning, prokaryotic expression andimmuneblotting study. The spore wall protein was successfully cloned of the gene encoding, and constructed of prokaryotic expression vector of pGEX4T-1/ALSWP1,and expressed in E. coli. Fusion proteins were purified by affinity chromatographyusing a GST-Tag Purification Kit. At the same time, the ALSWP1-specific polyclonalantibody was generated by immunizing rabbit with the purified fusion proteins andused in western blot analysis.The results clearly demonstrated that the preparedantiserum could be used to localization study.In order to further determine expression specificity and subcellular localizationcharacteristics of the protein during the different development stages of AntonosporaLocustae, intraeellular Parasites were examined by the indirect immunofluorescence(IFA) and immune electron microscopy (IEM). Both results of the two studies showedthat the putative ALSWP1is a spore wall protein.The expression pattern of the spore wall protein ALSWP1was presented in theinfected midgut of Locusta migratoria manilensis.The results indicate that the transcripts of β-tubulin could be detected at11d, and asthe infection time of growth, correspondingly the brightness of the band increasedsignificantly. Meanwhile, ALSWP1only can be detected at15d, and the transcripts stayat an obvious expression all the time.Using the recombinant fusion protein ALSWP1as the bait protein, using GSTprecipitation test to separate the host target proteins which have the interaction withALSWP1, and three hypothetical target interacting protein were identified with massspectrometry, preliminary results obtained with3putative interaction of the targetprotein.A3-dimensional model of ALSWP1was built by homology modeling with3M4F_A（14%）.