分枝杆菌噬菌体D29 LysinB/Holin真核表达载体的构建及杀菌试验  

Construction of eukaryotic expression vector for bacteriophage D29 LysinB/Holin and analysis of its bactericidal activity

作　　者：席志阳 宋通 王文涛 吴文潇 付玉荣 伊正君 Xi Zhiyang;Song Tong;Wang Wentao;Wu Wenxiao;Fu Yurong;Yi Zhengjun(School of Medical Laboratory,Shandong Second Medical University,Weifang 261053,China;School of Basic Medicine,Shandong Second Medical University,Weifang 261053,China)

机构地区：[1]山东第二医科大学医学检验学院,潍坊261053 [2]山东第二医科大学基础医学院,潍坊261053

出　　处：《中华微生物学和免疫学杂志》2024年第1期74-80,共7页Chinese Journal of Microbiology and Immunology

基　　金：山东省自然科学基金面上项目(ZR2021MH401)。

摘　　要：目的构建分枝杆菌噬菌体D29 LysinB/Holin融合蛋白真核表达载体,通过细胞感染模型研究其在胞内对结核分枝杆菌的杀伤效果。方法构建原核重组质粒pET32a-LysinB并诱导表达,纯化蛋白后制备多克隆抗体;构建真核重组质粒pcDNA3.1(+)-LysinB/Holin并转染进单核巨噬细胞RAW264.7,经制备的LysinB多克隆抗体进行表达鉴定后,建立细胞感染模型并通过抗酸染色和菌落计数检测LysinB/Holin融合蛋白的杀菌效果。结果成功制备LysinB的多克隆抗体。重组质粒pcDNA3.1(+)-LysinB/Holin在真核细胞中有效表达LysinB/Holin融合蛋白且对细胞无明显毒性。LysinB/Holin融合蛋白可有效杀伤胞内的结核分枝杆菌。结论重组质粒pcDNA3.1(+)-LysinB/Holin对胞内结核分枝杆菌有较好的杀伤效果,且对细胞无明显毒性,具有治疗结核病的潜能。Objective To construct a eukaryotic expression vector for bacteriophage D29 LysinB/Holin fusion protein and study its bactericidal efficacy against Mycobacterium tuberculosis(Mtb)in a cell infection model.Methods A recombinant plasmid pET32a-LysinB was constructed and induced to express LysinB.The polyclonal antibody against LysinB was prepared after the purification of LysinB.A recombinant plasmid pcDNA3.1(+)-LysinB/Holin was constructed and transfected into mononuclear macrophages RAW264.7.After the expression of the prepared polyclonal antibody was identified,a cell infection model was established and the bactericidal efficacy of LysinB/Holin fusion protein was measured by acid-fast staining and colony counting.Results The polyclonal antibody against LysinB was successfully prepared.The recombinant plasmid pcDNA3.1(+)-LysinB/Holin could effectively express LysinB/Holin fusion protein in eukaryotic cells without inducing significant cytotoxicity.LysinB/Holin fusion protein was effective in killing Mtb in cells.Conclusions The recombinant plasmid pcDNA3.1(+)-LysinB/Holin has a better killing effect on intracellular Mtb without inducing obvious cytotoxicity against eukaryotic cells,showing a potential in the treatment of tuberculosis.

关 键 词：结核分枝杆菌 噬菌体D29 Lysin B HOLIN 

分 类 号：R392-33[医药卫生—免疫学]