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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。
Objective To study the characteristics of transfection of peripheral blood mononuclear cells with the recombinant eukaryotic expression plasmid 85A (antigen85A, Ag85A) of human Mycobacterium tuberculosis 85A (pcDNA3.1-Ag85A) to investigate whether the target gene Ag85A can be expressed in monocytes, Immunocompetent human peripheral blood mononuclear cells. Methods Monocytes were isolated from human peripheral blood mononuclear cells (PBMCs) and cultured in vitro. The purity of mononuclear cells was identified by flow cytometry. Ag85A cells were transfected with pcDNA3.1-Ag85A recombinant plasmid. Ag85A was detected by RT- The expression of target protein Ag85A was identified by Western blot. T lymphocytes were isolated from peripheral blood and co-incubated with the transfected mononuclear cells. The empty plasmid transfection group and the recombinant plasmid + monocyte were used as the control group. After 3 days, the proliferation of T lymphocytes was detected by MTT assay . Results A large number of high purity monocytes were obtained from primary cultured human monocytes after 6 days of primary culture. Flow cytometry confirmed the high expression of CD14 positive cells (98.35%) on human monocytes. RT-PCR The identification of Ag85A gene showed that the Ag85A gene was successfully transcribed in the transfected monocytes. Western blot confirmed that the transfected monocytes could synthesize the target protein Ag85A. After incubation with peripheral blood T lymphocytes, the monocytes transfected with the recombinant plasmids had stronger ability of stimulating the proliferation of T lymphocytes than the control group. Conclusion Ag85A recombinant eukaryotic expression plasmid pcDNA3.1-Ag85A was successfully transfected into human peripheral blood mononuclear cells, the target gene Ag85A expression in human monocytes. Transfection of peripheral blood mononuclear cells can stimulate peripheral blood T lymphocyte proliferation, thus providing a new way for the development of vaccine against tuberculosis and the treatment of TB patients with acquired immunodeficiency disease.