Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.
Evaluation of procedures for outer membrane isolation from Campylobacter jejuni.
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DOI：
10.1099/mic.0.024539-0
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发表时间：
2009-03
期刊：
影响因子：
--
通讯作者：
Thompson SA
中科院分区：
文献类型：
--
作者：
Hobb RI;Fields JA;Burns CM;Thompson SA
尽管空肠弯曲菌感染是全球胃肠炎的主要病因之一，但对于引发保护性免疫反应所需的因素，人们了解相对较少。研发针对这种病原体的疫苗的必要性已得到广泛认可，并且已经对多种候选疫苗进行了测试，取得了不同程度的成果；然而，目前仍缺乏合适的疫苗。为了更好地了解该菌的外膜蛋白成分，需要一种“金标准”方法来纯化外膜。因此，我们尝试开发一种稳健且可靠的方法，以获得纯净的外膜组分。我们共研究了九种方法，并通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）以及使用细胞质、细胞质膜和外膜的亚细胞标记物进行免疫印迹分析。我们发现，甘氨酸提取法、使用曲拉通X - 100的差异去污剂提取法、使用1M Tris（pH 7）的连续提取法、溶菌酶和超声处理的原生质球制备法以及碳酸盐提取法，均无法制备出纯净的外膜制剂。不过，我们确定了三种能提供无亚细胞污染的外膜组分的方法。使用30% - 60%蔗糖梯度的等密度离心法产生了七个无细胞质或细胞质膜污染的组分；然而，这些组分与典型的外膜相关峰（如大肠杆菌或沙门氏菌的外膜峰）的对应程度不如预期。仅使用溶菌酶的原生质球法以及对该样品进行碳酸盐洗涤，也能得到纯净的外膜组分。使用N - 月桂酰肌氨酸（十二烷基肌氨酸钠，Sarkosyl）提取外膜，得到的样品最纯净且重现性最佳。这些外膜制剂将有助于未来旨在鉴定空肠弯曲菌表面蛋白作为疫苗成分的研究。
Although infection with Campylobacter jejuni is one of the leading causes of gastroenteritis worldwide, relatively little is known about the factors that are required to elicit a protective immune response. The need for a vaccine against this pathogen is well recognized and a number of vaccine candidates have been tested with varying degrees of success; however, there is still a lack of a suitable vaccine. To gain a better understanding of the outer-membrane protein components of this organism, a ‘gold standard’ method to purify the outer membrane is needed. Therefore, we attempted to develop a robust and reliable method which resulted in a pure outer-membrane fraction. A total of nine methodologies were examined and analysed by SDS-PAGE and immunoblotting using subcellular markers for the cytoplasm, cytoplasmic membrane and outer membrane. We found that glycine extraction, differential detergent extraction using Triton X-100, serial extraction using 1 M Tris pH 7, spheroplasting by lysozyme and sonication, and carbonate extraction did not produce pure outer-membrane preparations. However, we identified three methods that provided outer-membrane fractions free from subcellular contamination. Isopycnic centrifugation using a 30–60 % sucrose gradient produced seven fractions free from cytoplasmic or cytoplasmic membrane contamination; however, these fractions did not correspond as well as expected with the typical outer-membrane-associated peak (e.g. Escherichia coli or Salmonella). The spheroplast method using lysozyme alone also resulted in pure outer-membrane fraction, as did carbonate washing of this sample. The extraction of outer membranes using N-lauroylsarcosine (Sarkosyl) produced the purest and most reproducible sample. These outer-membrane preparations will be useful for future studies aimed at identifying C. jejuni surface proteins as vaccine components.
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影响因子：
3.2
作者：
HANCOCK, REW;NIKAIDO, H
通讯作者：
NIKAIDO, H
CetA 和 CetB（空肠弯曲杆菌中的双向能量趋向系统）的表征。
影响因子：
3.6
作者：
Elliott KT;Dirita VJ
通讯作者：
Dirita VJ
影响因子：
3.1
作者：
Albert, M. John;Haridas, Shilpa;Adler, Ben
通讯作者：
Adler, Ben
影响因子：
3.5
作者：
Dé, E;Jullien, M;Bolla, JM
通讯作者：
Bolla, JM
DOI：
10.1007/bf01962343
发表时间：
1988-06-01
影响因子：
4.5
作者：
HERBRINK, P;VANDENMUNCKHOF, HAM;VANDIJK, WC
通讯作者：
VANDIJK, WC