Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000
Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000
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DOI：
10.1186/1475-2859-7-24
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发表时间：
2008-07-29
影响因子：
6.4
通讯作者：
Gahan, Cormac G. M.
中科院分区：
文献类型：
--
作者：
Bahey-El-Din, Mohammed;Griffin, Brendan T.;Gahan, Cormac G. M.
背景：单核细胞增生李斯特菌是一种典型的食源性致病菌，感染孕妇和免疫功能低下的个体。李斯特菌溶血素O（LLO）是李斯特菌的主要毒力因子，常被用作检测李斯特菌的诊断标志物。单核细胞增多症此外，LLO代表在感染期间驱动T细胞介导的免疫的有效抗原。在本工作中，使用乳酸乳球菌NZ 9000作为表达宿主，使用NICE（NIsin控制表达）系统在诱导条件下超生产LLO。结果：构建的表达载体pNZPnisA：CYTO-LLO在L.乳酸链球菌NZ 9000的最佳诱导时间为对数中期，在30 ℃下用0.2%v/v乳链菌肽静态诱导4 h。通过Ni-NTA亲和层析完成His标记的LLO的纯化，并通过溶血测定确认功能性。总LLO产量（以总蛋白含量测量）为4.43-5.9 mg/L培养物，并且在4 ℃下储存8个月后仍可检测到溶血活性。结论：本工作中描述的LLO生产方法提供了一种在革兰氏阳性乳球菌中有效生产LLO的方法，以产生用于研究和诊断应用的蛋白质的重要来源。LLO在L. lactis比E.这可能有助于该系统在体内和体外的应用。
Background: Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter.Results: The constructed vector (pNZPnisA:CYTO-LLO) was expressed in L. lactis NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30 degrees C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.43-5.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4 degrees C.Conclusion: The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system.