结核杆菌Hsp65与EGFP融合基因的构建及DC疫苗的制备

目的: 构建结核杆菌H37Rv株Hsp65与增强型绿色荧光蛋白 (EGFP)的融合基因pEGHsp65, 并以其转染小鼠的树突状细胞 (DC), 制备抗结核的DC疫苗。方法: 采用PCR技术, 从培养的结核杆菌H37Rv株中抽提Hsp65基因,克隆到含有EGFP基因的质粒pEGFP- C1中, 构建pEGHsp65融合基因。以其转染Hela细胞, 在共聚焦激光扫描荧光显微镜下观测不同时间荧光表达的强弱, 并用RT- PCR检测Hsp65mRNA的表达。以pEGHsp65融合基因转染小鼠骨髓细胞经GM- CSF和IL -4诱导分化的DC, 用MTT比色法检测DC疫苗刺激未致敏脾细胞的增殖。结果: 用EcoRⅠ和BglⅡ双酶切鉴定证实, H37Rv株Hsp65DNA已插入重组表达载体pEG -FP- C1中。将融合基因转染入Hela细胞, 48h转染率最高;用RT- PCR在mRNA水平上可检测到Hsp65mRNA的表达。用MTT比色法检测表明, 融合基因转染的DC能激活并引起未致敏的脾细胞增殖。结论: 成功地构建pEGHsp65融合基因和以其制备的DC疫苗, 为进一步观察其治疗结核病的效应奠定了基础。

Construction of the fusion gene of Hsp65 of Mycobacterium tuberculosis and EGFP and preparation of dendritic cell vaccine against tuberculosis

AIM: To construct the fusion gene of Hsp65 of Mycobacterium tuberculosis H37Rv and enhanced green fluorescence protein (EGFP) and prepare dendritic cell (DC) vaccine. METHODS: Hsp65 DNA amplified by PCR was cloned into eukaryotic expression vector EGFP-C1. The recombinant plasmid pEGHsp65 was subsequently transfected into Hela cells, and the transfection rate was determined under confocal laser scanning fluorescence microscope at different times. RT-PCR was used to detect the expression of Hsp65 mRNA in Hela cells. The GM-CSF and IL-4 induced DCs from mouse bone marrow cells were transfected with recombinant plasmid pEGHsp65. Proliferation of unprimed splenocytes activated by transfected DCs was detected by MTT colorimetry. RESULTS: Restrictive enzyme digestion analysis (EcoR I, Bgl II) confirmed that Hsp65 DNA had been inserted into the vector pEGFP-C1. The recombinant plasmid pEGHsp65 was transfected into Hela cells and the expression of the fusion gene reached peak at the 48 hours after transfection. Expression of Hsp65 mRNA was detected in Hela cells by RT-PCR. DCs transfected with pEGHsp65 could stimulate the proliferation of unprimed splenocytes. CONCLUSION: The pEGHsp65 fusion gene was successfully constructed and DCs transfected with the pEGHsp65 might be a candidate vaccine for tuberculosis.