Construction of eukaryotic expression vector for bacteriophage D29 LysinB/Holin and analysis of its bactericidal activity
10.3760/cma.j.cn112309-20230426-00111
- VernacularTitle:分枝杆菌噬菌体D29 LysinB/Holin真核表达载体的构建及杀菌试验
- Author:
Zhiyang XI
1
;
Tong SONG
;
Wentao WANG
;
Wenxiao WU
;
Yurong FU
;
Zhengjun YI
Author Information
1. 山东第二医科大学医学检验学院，潍坊 261053
- Keywords:
Mycobacterium tuberculosis;
Bacteriophage D29;
LysinB;
Holin
- From:
Chinese Journal of Microbiology and Immunology
2024;44(1):74-80
- CountryChina
- Language:Chinese
-
Abstract:
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.
