三种李斯特菌菌体蛋白提取方法的比较

Comparison of Three Methods for Extraction of Non-secreted Proteins from Listeria

通过对3种李斯特菌菌体蛋白提取方法主要是破壁方式的比较,找到一种可高效提取李斯特菌菌体蛋白的方法,为该菌的深入研究提供可靠技术。以绵羊李斯特菌新鲜液体培养物为材料,收集菌沉淀,分别选用3种破壁方式破除细胞壁,三氯乙酸(trichloroacetic acid,TCA)-丙酮沉淀法沉淀破壁液中的菌体蛋白,采用聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)和Western-blot分析所得菌体蛋白。溶菌酶-超声破壁-TCA-丙酮沉淀法提取得到的李斯特菌菌体蛋白SDS-PAGE条带清晰丰富,Western-blot条带特异。溶菌酶-超声破壁-TCA-丙酮沉淀法可有效提取李斯特菌菌体蛋白,满足常用蛋白分析技术的要求,是一种提取李斯特菌菌体蛋白的可靠方法。

Three methods of extracting non-secreted proteins from Listeria were compared, mainly different in lysis process, to obtain an efficient non-secreted proteins extracting method for Listeria, to provide a reliable technique for the further study of Listeria. Fresh broth culture of Listeria ivanovii was used as study material. Bacteria deposit was collected by centrifugation, and three lysing approaches were adopted sepa-rately. Non-secreted proteins were obtained through the method of trichloroacetic acid（TCA）-acetone pre-cipitation in the lysis solution, and verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis（SDS-PAGE） and Western-blot. SDS-PAGE bands of non-secreted proteins extracted via the method of lysozyme-ultrasonication-TCA-acetone precipitation were distinct and sufficient, and the Western-blot re-sults were specific. The method of lysozyme-ultrasonication-TCA-acetone precipitation can be used to ex-tract Listeria non-secreted proteins efficiently, which is qualified for common protein experiments, and is a reliable method for extracting non-secreted proteins from Listeria.