CAJ | 학술논문

目的构建携带人颗粒溶素(GLS)的真核表达质粒并探讨其表达后对巨噬细胞RAW264.7内结核分枝杆菌的杀菌能力.方法利用套式PCR从同种异型抗原激活的人CTL中扩增出GLS基因,并克隆入pBudCE4.1载体,构建重组质粒.将其转染至感染结核分枝杆菌H37Rv的巨噬细胞株RAW264.7中,套式PCR、免疫荧光检测GLS表达;转染96 h后裂解细胞作抗酸染色及菌落计数,检测GLS细胞内直接杀菌活性.数据行t或t'检验.结果成功构建携带GLS的真核表达质粒pBudCE4.1/GLS;在转染的已感染H37Rv巨噬细胞株RAW264.7中,从转录和翻译水平都检测到目标基因GLS的表达产物;转染96 h,佛波酯+pBudCF4.1/GLS组、佛波酯+pBudCE4.1组与未激活初始巨噬细胞荷菌量分别为(1.44±1.25)、(3.16±0.20)和(3.59±0.21)个,两两比较,差异均有统计学意义(t=2.403,t=2.854,均P＜0.05).结论携带人GLS的真核表达质粒在巨噬细胞表达后具有明显的杀菌活性,为进一步作为结核基因治疗疫苗的应用提供了实验依据.
목적 구건휴대인과립용소(GLS)적진핵표체질립병탐토기표체후대거서세포RAW264.7내결핵분지간균적살균능력.방법이용투식PCR종동충이형항원격활적인CTL중확증출GLS기인,병극륭입pBudCE4.1재체,구건중조질립.장기전염지감염결핵분지간균H37Rv적거서세포주RAW264.7중,투식PCR、면역형광검측GLS표체;전염96 h후렬해세포작항산염색급균락계수,검측GLS세포내직접살균활성.수거행t혹t'검험.결과성공구건휴대GLS적진핵표체질립pBudCE4.1/GLS;재전염적이감염H37Rv거서세포주RAW264.7중,종전록화번역수평도검측도목표기인GLS적표체산물;전염96 h,불파지+pBudCF4.1/GLS조、불파지+pBudCE4.1조여미격활초시거서세포하균량분별위(1.44±1.25)、(3.16±0.20)화(3.59±0.21)개,량량비교,차이균유통계학의의(t=2.403,t=2.854,균P＜0.05).결론휴대인GLS적진핵표체질립재거서세포표체후구유명현적살균활성,위진일보작위결핵기인치료역묘적응용제공료실험의거.
Objective To construct eukaryotic expression recombinant plasmid containing human granulysin(GLS) and investigate the effect of GLS expression in macrophage RAW264.7 cells on the bactericidal activity against intracellular Mycobacterium tuberculosis.Methods GLS gene was amplified by nested-polymerase chain reaction(PCR) from human cytotoxicity T lymphocyte(CTL) activated by allogenic antigen,and inserted into pBudCE4.1 vector to construct recombinant plasmid.Subsequently,the plasmid was transfccted into RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The expression of GLS was detected by nested-PCR and immunocytochemistry method.The RAW264.7 cells were lysed after transfected for 96 h,then acidfast stained,cultivated and colony count were done to determine the intraeellular bactericidal activity of GLS.The data were analyzed by t or t' test.Results The pBudCE4.1/GLS eukaryotic expression recombinant plasmid was successfully constructed.The transcriptional and translational expressions of target gene GLS were detected in RAW264.7 cells which were infected with Mycobacterium tuberculosis H37Rv.The bacterial load in macrophages of phorbol myristate acetate(PMA)+pBudCE4.1/GLS group,PMA+pBudCEA.1 group and non-activated group were 1.44±1.25,3.16±0.20 and 3.59±0.21,respectively.The differences between groups were all significant (t=2.403,t=2.854,both P＜0.05).Conclusion Eukaryotic expression recombinant plasmid carrying human GLS gene expressed in macrophages has strong bactericidal activity against intracellular mycobacteria,which provide information for the further study on therapeutic vaccine against Mycobacterium tuberculosis.