Establishing Proteomic Platform Of 2-D Electrophoresis And Mass Spectrum Analyses Of Nosema Bombycis

Microsporidia are obligate intracellular parasites that can infect a wide variety of animals ranging from insects to human. Nosema bombyds is the pathogen of Pebrine, that can cause a big death to silkworm due to vertical and horizontal transmission, and it is one of big threats for the whole silkworm industry. The first assignment of Nosema bombycis proteome is to analyze the total proteins, the widely used technologies include 2-D electrophoresis, Mass Spectrum. Here we report the establishment of 2-D electrophoresis and Mass spectrum analyses of total proteins of mature spores of Nosema bombyds, the result is below:1. The preparation of the total proteins of mature spores. The ways to extract protein of Nosema bombyds includes chemical and physical approaches. The chemical method is using the 0.1mol/L K₂CO₃ to trigger the germination of spores, the proteins of the released sporoplasma can be easy to obtain treated by the lysis solution, However, this method can bring some salt ions to the protein smples, which is troublesome in 2-D electroporesis. The another is physical method, it mixs the mature spores and liquid nitrogen to grind, and uses lysis solution to extract proteins, the process is a little complicated but can collect abundant proteins with little salt ions, which is suitable for 2-D electrophoresis.2. The referencial 2-DE map of Nosema bombycis. We have obtained a complete referencial map of Nosema bombyds, which contains proteins with molecular weight mainly less 45kD, and spreading in different gradient of pH of the gel. Low molecular weight proteins are abundant, while the high ones are in low quatity, especially the high ones in the regions of alkalescent pH.3. Proteins identification with peptide mass fingerprinting by MALDI-TOF-MS. We set up the protein database according to the predicted coding sequence based on the whole genome database of Nosema bombycis. 50 protein spots have been identified by MALDI-TOF-MS, which include 28 proteins with known function, 9 hypothetical proteins and 13 unknown proteins without any function information available. These proteins are divided by several groups according to their functions, including protein destination and degradation, transcripition, transposon, cellular organization and biogenesis, cell rescue, defense , cell death and ageing, cell growth and division and DNA synthesis, cellular communication/signal transduction, protein synthesis and so on, which involved nearly all the essential physiological activitys. The furthermore, we have found some interesting proteins such as: polar tube protein 2, surface-antigen protein P30.4. thioltransferase (glutaredoxin), vcCyP , thioredoxin reductase and proteins participated the transposon activity.In conclusion: We have obtain a complete proteomics picture of Nosema bombycis according to the SDS-PAGE, 2-D electrophoresis and MALDI-TOF-MS, and 50 protein spots were identified, some of which are related to significant physiological process. These researches have established a comprehensive platform for proteomic research of Nosema bombycis.