Differential Proteomics Analysis Of Four Microsporidia And The Prokaryotic Expression Of NbSwp7

Microsporidia are unicellular eukaryotes and obligate intracellular parasites. The host rangeis extremely wide extending from invertebrates to vertebrates. Microsporidia are of multiplespecies, and they can spread horizontally and vertically. The pathogen brings incalculableeconomic losses to the all sericulture countries in the world.The gene evolution of microsporidia is faster than genome evolution, therefore, theresearches on microsporidia transfer from the gene to protein level while entering to thepost-genomic era. Many studies suggested that the spore protein especially spore wallprotein and polar tube protein played an important role in host infection. Proteomics hasbecome an important field studying microsporidia. The study analyzed the differentproteomics between Nosema bombycis and Nosema sp. HA, Nosema sp. PA,Endoreticulatus sp. Zhenjiang by using two-dimensional electrophoresis and identified thedifferent protein spots with matrix-assisted laser desorption-ionization of flight-time massspectrometry.The results indicated that, the total protein distributions of Nosema bombycis and Nosema sp.HA, Nosema sp. PA are smiliar. Comparing with Nosema bombycis, Nosema sp. HA mainlylacked a spot named PTP3and Nosema sp. PA mainly lacked multiple PTP3spots and oneNbSwp5spot which both related to spore structural integrity and host infection. The threemicrosporidia were of the same genus but they possed different virulence strength tosilkworm, which suggested that the polar tube and spore wall protein of the latter two sporesmight have different molecular sizes and/or structures from Nosema bombycis. It waspredicted that the PTP3of Nosema sp. HA was slightly different from Nosema bombycisand Nosema sp. HA lacked PTP3and Swp5or the two proteins of both two microsporidiawere of lower homology. In addition, there were some other different proteins whichpossibly affected the life activities of microsporidia such as parasite and spreading processesthrough pathways of energy metabolism, electron transfer, material transport, proteinmodification, DNA helicase and repair and so on.The comparative proteomic analysis of Nosema bombycis and Endoreticulatus sp.Zhenjiang indicated that their total protein distributions were almost completely different.Comparing with Nosema bombycis, Endoreticulatus sp. Zhenjiang mainly lacked PTP3,various kinds of spore wall proteins of Nosema bombycis, ATPase and manganesesuperoxide dismutase which respectively involved in spore structural integrity, host infection, energy metabolism and maintaining the concentration balance of superoxide ion.Conversely, Nosema bombycis mainly lacked transcription initiation factor IIA,ATP-dependent RNA helicase, zinc-dependent aminopeptidase, DNA J-class molecularchaperone，translation elongation factor and ATPase which respectively involved in theregulation of spore transcription, translation, energy metabolism, protein degradation andmaintaining cell stability and so on. The two microsporidia belonged to different genus andspecies and they also possed different virulence strength to silkworm, which suggested thatthe proteins of Endoreticulatus sp. Zhenjiang had different molecular sizes, classes and/orstructures from Nosema bombycis or they were of very lower homology. Endoreticulatus sp.Zhenjiang was predicted to have no PTP3or spore wall proteins with typical structure inNosema bombycis, which affected infectivity to silkworm. In addition, there were someother different proteins which possibly affected the life activities of microsporidia throughthe regulation of gene and/or protein level.Nosema bombycis had many spore wall proteins in which NbSwp7the study on what wasless was successfully identified with proteomics technology. The encoding gene of NbSwp7that sized876bp was successfully obtained through database search and using the tools ofgene cloning, nucleotide sequencing detection means and so on. Then the recombinantplasmid was successfully builded with PET-30a vector and E. coli competent cells usingenzyme digestion, ligation and transformation. At last, the expression was completed with1μmol/mL IPTG at30oС for5h. SDS-PAGE and western blot indicated that the size ofexpression products was slightly larger than the predicted value, which was caused byprotein structure change owing to the translation and post-translational modificationprocesses.In the study, we discovered that it existed differences between various kinds ofmicrosporidia, especially PTP and spore wall protein that related to host invasion. The studynot only laid foundation for the depth research of the various terms of microsporidiaproteins such as the classes, structures, functions, localizations and so on, but also providedevidences for the research on diagnosis of pebrine disease in future.