基于厌氧条件下转录组分析的单增李斯特氏菌强启动子的鉴定

Identification of strong promoters from Listeria monocytogenes based on RNA-seq analysis under anaerobic conditions

减毒单增李斯特氏菌是一种有潜力的肿瘤疫苗载体和靶向药物载体。然而由于缺乏在肿瘤缺氧环境中调控外源蛋白表达量的基因元件,限制了减毒单增李斯特氏菌活载体的进一步发展。本研究通过对单增李斯特氏菌EGD-e菌株进行转录组分析,在厌氧和有氧两种培养条件下,筛选出18个候选强启动子并以此构建了绿色荧光蛋白报告菌株,然后根据菌株的荧光强度从中选出了调控表达水平最高的P_(an)4启动子。此外,P_(an)4启动子还可成功调控天青蛋白和θ毒素表达,具有一定的广泛适用性。本研究为减毒单增李斯特氏菌在肿瘤药物或疫苗活载体应用中外源蛋白表达调控的优化提供了有效的分子工具。

Attenuated L.monocytogenes is a potential tumor vaccine carrier and targeted drug carrier.However,the lack of genetic elements that regulate the expression of foreign proteins in the hypoxic environment of tumors limited the further development of live attenuated L.monocytogenes vectors.In this study,RNA-seq analysis of L.monocytogenes EGD-e strain was carried out.Under anaerobic and aerobic culture conditions,eighteen promoters were screened and the green fluorescent protein report strains were constructed.The P4 promoter with the highest regulatory expression level was selected according to the fluorescence intensity of the strain.In addition,the P4 promoter could also successfully regulate the expression of azurin andθtoxin,which had a certain broad applicability.This study provided an effective molecular tool for optimizing the expression and regulation of exogenous proteins in the application of attenuated L.monocytogenes in tumor drugs or vaccine live vectors.