单核细胞增生李斯特菌1/2a和4b血清型中李斯特菌溶血素O的免疫亲和纯化、稳定化及比较表征

Immunoaffinity purification, stabilization and comparative characterization of listeriolysin O from Listeria monocytogenes serotypes 1/2a and 4b.

作者信息

Matar G M, Bibb W F, Helsel L, Dewitt W, Swaminathan B

机构信息

Division of Bacterial and Mycotic Diseases, Centers for Disease Control, Atlanta, GA 30333.

出版信息

Res Microbiol. 1992 Jun;143(5):489-98. doi: 10.1016/0923-2508(92)90095-6.

DOI:10.1016/0923-2508(92)90095-6

PMID:1448624

Abstract

We developed a simple and highly effective procedure for stabilizing the haemolytic activity of listeriolysin O (LLO) from Listeria monocytogenes after immunoaffinity purification. The haemolytic activity of LLO was stabilized by eluting it directly into tubes containing an alkaline buffer (5 mM lysine, 140 mM KCl, 50% ethylene glycol, pH 11.5). The purified LLO retained 100% of its haemolytic activity after 6 weeks of storage at -20 degrees C. LLO purified from a strain of L. monocytogenes serotype 1/2a (ATCC 43249) and LLO purified from a strain of L. monocytogenes serotype 4b (F 2365) isolated from a Mexican-style cheese, showed no significant differences in pH and temperature stability. When incubated in buffers at pH values from 4 to 12 at 4 degrees C and 25 degrees C, LLO from serotypes 1/2a and 4b retained maximal haemolytic activity at pH 8 after 4 h of incubation. LLO from both serotypes lost their haemolytic activity after incubation at 50 degrees C for 25 min.

摘要

我们开发了一种简单且高效的方法，用于在免疫亲和纯化后稳定单核细胞增生李斯特菌中李斯特菌溶血素O（LLO）的溶血活性。通过将LLO直接洗脱到含有碱性缓冲液（5 mM赖氨酸、140 mM KCl、50%乙二醇，pH 11.5）的试管中，可稳定其溶血活性。纯化后的LLO在-20℃储存6周后仍保留100%的溶血活性。从单核细胞增生李斯特菌血清型1/2a菌株（ATCC 43249）纯化的LLO和从墨西哥风味奶酪中分离出的单核细胞增生李斯特菌血清型4b菌株（F 2365）纯化的LLO，在pH值和温度稳定性方面无显著差异。当在4℃和25℃下于pH值4至12的缓冲液中孵育时，血清型1/2a和4b的LLO在孵育4小时后于pH 8时保留最大溶血活性。两种血清型的LLO在50℃孵育25分钟后均失去溶血活性。