摘要
试验旨在研究结核分枝杆菌Rv2626c蛋白对小鼠巨噬细胞RAW264.7细胞凋亡的影响。根据GenBank数据库中结核分枝杆菌Rv2626c基因序列设计引物,并以结核分枝杆菌国际标准株H37Rv cDNA为模板,PCR扩增Rv2626c基因并克隆至慢病毒表达载体pLEX-EGFP中,包装慢病毒并感染RAW264.7细胞,使用Western blotting和流式细胞仪技术检测Rv2626c蛋白表达水平和RAW264.7细胞凋亡率变化。结果显示,成功构建慢病毒表达载体pLEX-EGFP-Rv2626c;成功包装慢病毒并感染RAW264.7细胞;Rv2626c蛋白在RAW264.7细胞中高水平表达显著促进了细胞凋亡。本试验结果表明,在RAW264.7细胞中过表达结核分枝杆菌Rv2626c蛋白能显著性增加其凋亡水平。
The assay was aimed to study the effects of Mycobacterium tuberculosis Rv2626 cprotein on RAW264.7cell apoptosis.According to the Rv2626 c gene sequence of Mycobacterium tuberculosis in GenBank database,Rv2626 cprimers were designed.The Rv2626 c gene was amplified and cloned into the lentiviral vector pLEX-EGFP.After transfection of lentivirus expressing vector with the helper plasmids into HEK-293 Tcells,lentivirus were packaged and infected into RAW264.7cells.Then total protein of the infected cells was extracted.Protein levels of Rv2626 c and apoptosis levels of RAW264.7cells were detected using Western blotting and flow cytometry.The results showed that reconstructed lentivirus expressing vector pLEX-EGFP-Rv2626 cand the Rv2626c-overexpressing lentivirus Rv2626c-lv were constructed successfully.The overexpressed Rv2626 cprotein promoted the RAW264.7cells apoptosis.Overexpression of Mycobacterium tuberculosis Rv2626 cprotein significantly promoted apoptosis in RAW264.7cells.
出处
《中国畜牧兽医》
CAS
北大核心
2016年第4期892-898,共7页
China Animal Husbandry & Veterinary Medicine
基金
国家科技重大专项项目(2013BAI05B05
2013ZX10003003-002)
