布鲁氏菌侵染对类泛素SUMO-1表达的影响及类泛素SUMO-1慢病毒表达载体的构建

Effect on Expression of SUMO-1 in Cells Infected by Brucellaand Construction of Lentivirus Vector that Over-expressing SUMO-1

研究布鲁氏菌侵染小鼠巨噬细胞RAW264.7后类泛素SUMO-1的表达变化,并构建小鼠类泛素SUMO-1基因过表达的慢病毒载体。本试验分别利用布鲁氏菌16M、M5-90侵染小鼠巨噬细胞RAW264.7,利用实时荧光定量PCR和Western blotting分别检测细胞中类泛素SUMO-1的表达;利用DNA重组技术将类泛素SUMO-1基因片段插入到慢病毒表达载体pLEX-MCS中,获得重组慢病毒质粒pLEX-SUMO-1,测序鉴定成功后转染293T细胞,包装好的慢病毒感染小鼠巨噬细胞RAW264.7,并利用实时荧光定量PCR、Western blotting方法分别检测细胞中类泛素SUMO-1mRNA及蛋白表达水平。结果表明,布鲁氏菌16M、M5-90侵染细胞后,在感染早期类泛素SUMO-1的表达受到抑制,12h内呈现下降趋势,在12h后开始上升,极显著高于正常水平(P<0.01);测序结果证明,类泛素SUMO-1基因正确插入到pLEX-MCS质粒中;实时荧光定量PCR方法检测表明,类泛素SUMO-1mRNA转录水平上调。由此可见,布鲁氏菌侵染小鼠巨噬细胞RAW264.7能够引起细胞内类泛素SUMO-1表达的改变;成功构建了类泛素SUMO-1慢病毒过表达载体,为进一步研究类泛素SUMO-1的功能奠定基础。

To investigate the changes of SUMO-1（small ubiquitin-related modifier）gene in RAW264.7cells infected by Brucella,a lentiviral vector for the over expression of mouse SUMO-1was constructed.RAW264.7cells was infected by Brucella16 Mor M5-90,then quantitative Realtime PCR and Western blotting were used to detect SUMO-1in RAW264.7cells.SUMO-1gene fragment was inserted into the lentiviral vector pLEX-MCS in the use of recombinant,resulting into a recombinant lentivirus pLEX-SUMO-1.Recombinant lentiviral vector was transfected into293 Tcells after sequencing correctly,RAW264.7was infected by supernatant.Quantitative Realtime PCR and Western blotting were used to quantify the transcription of SUMO-1gene.The results showed that RAW264.7cells was infected by Brucella 16 M or M5-90,SUMO-1showed a reduced trend in 12 h,after 12 h,SUMO-1began to rise and became higher than normal levels,and the difference was extremely significant（P〈0.01）.The sequencing results showed that mouse SUMO-1gene lentiviral vector was constructed successfully.The mRNA expression of SUMO-1gene increased.The results indicated that the expression of SUMO-1changed after Brucellainfec-tion.Over-expression vector targeting SUMO-1gene was constructed successfully,an experimental basis on the SUMO-1functional study was provided.