Ag85A对人单核细胞活性和功能的影响

目的研究结核分枝杆菌分泌抗原85A(antigen85A,Ag85A)重组真核表达质粒pcDNA3.1-Ag85A转染人外周血单核细胞的特性,探讨目的基因Ag85A能否在单核细胞表达,从而制备具有免疫活性的人外周血单核细胞。方法分离人外周血单核细胞并进行原代培养,流式细胞仪鉴定单核细胞纯度;Ag85A重组真核表达质粒pcDNA3.1-Ag85A转染单核细胞;RT-PCR检测目的基因Ag85A在单核细胞中的表达,目的蛋白Ag85A的表达采用Western blot鉴定。从外周血分离得到T淋巴细胞,与转染后的单核细胞共同孵育,并设空质粒转染组、重组质粒+单核细胞为对照组,3 d后用MTT法检测T淋巴细胞增殖情况。结果人外周血单核细胞经原代培养6 d后,获得大量高纯度的单核细胞,流式细胞术鉴定人单核细胞表面特异性标记CD14阳性的细胞高表达(98.35%);RT-PCR法鉴定表明Ag85A基因在转染单核细胞中成功转录,Western blot证实转染后的单核细胞可合成目的蛋白Ag85A。与外周血T淋巴细胞共同孵育后,经转染重组质粒的单核细胞相对对照组,有更强的刺激T淋巴细胞增殖的能力。结论 Ag85A重组真核表达质粒pcDNA3.1-Ag85A成功转染人外周血单核细胞,目的基因Ag85A能在人单核细胞中的表达。经转染的外周血单核细胞有刺激外周血T淋巴细胞增殖能力,从而为结核病的疫苗的研制以及为患获得性免疫缺陷病的结核病人的治疗提供新途径。

Effects of Ag85A on the activity and function of human monocytes

This study is aimed to investigate the characteristics of the human monocytes transfected with the recombinant plasmid pcDNA3.1-Ag85A,and to study the expression of Ag85A gene and protein in human monocytes,for gaining immunocompetent monocytes.Monocytes were collected from human peripheral blood mononuclear cells(PBMC) and primarily cultured in vitro for 6 days,and then flow cytometry demonstrated a large number of adherent monocytes,highly expressed CD14,at about 98.35%.Subsequently,the monocytes were tra...