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

doi:10.16431/j.cnki.1671-7236.2016.06.004

›› 2016, Vol. 43 ›› Issue (6): 1422-1429.doi: 10.16431/j.cnki.1671-7236.2016.06.004

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

李启峰¹, 丁金花¹, 窦文静², 张辉¹, 陈创夫¹

1. 石河子大学动物科技学院, 石河子 832003;
2. 石河子市兽医卫生检疫所, 石河子 832000

收稿日期:2015-11-18 出版日期:2016-06-20 发布日期:2016-07-11
通讯作者: 陈创夫 E-mail:ccf-xb@163.com
作者简介:李启峰(1989-),男,新疆奎屯人,硕士生,研究方向:布鲁氏菌致病机制,E-mail:604560953@qq.com
基金资助:
国家自然科学基金(31260596、31572491)

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

LI Qi-feng¹, DING Jin-hua¹, DOU Wen-jing², ZHANG Hui¹, CHEN Chuang-fu¹

1. College of Animal Science and Technology, Shihezi University, Shihezi 832003, China;
2. Shihezi Veterinary Health Quarantine Institute, Shihezi 832000, China

Received:2015-11-18 Online:2016-06-20 Published:2016-07-11

摘要/Abstract

摘要： 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-1 mRNA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,12 h内呈现下降趋势,在12 h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-1 mRNA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。

关键词: 布鲁氏菌; 类泛素SUMO-1; 慢病毒载体; 小鼠巨噬细胞

Abstract: To investigate the changes of SUMO-1 (small ubiquitin-related modifier) gene in RAW264.7 cells infected by Brucella,a lentiviral vector for the over expression of mouse SUMO-1 was constructed.RAW264.7 cells was infected by Brucella 16M or M5-90,then quantitative Real-time PCR and Western blotting were used to detect SUMO-1 in RAW264.7 cells.SUMO-1 gene 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 into 293T cells after sequencing correctly,RAW264.7 was infected by supernatant.Quantitative Real-time PCR and Western blotting were used to quantify the transcription of SUMO-1 gene.The results showed that RAW264.7 cells was infected by Brucella 16M or M5-90,SUMO-1 showed a reduced trend in 12 h,after 12 h,SUMO-1 began to rise and became higher than normal levels,and the difference was extremely significant (P < 0.01).The sequencing results showed that mouse SUMO-1 gene lentiviral vector was constructed successfully.The mRNA expression of SUMO-1 gene increased.The results indicated that the expression of SUMO-1 changed after Brucella infection.Over-expression vector targeting SUMO-1 gene was constructed successfully,an experimental basis on the SUMO-1 functional study was provided.

Key words: Brucella; small ubiquitin-related modifier SUMO-1; lentivirus vector; murine macrophages

中图分类号:

李启峰, 丁金花, 窦文静, 张辉, 陈创夫. 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[J]. , 2016, 43(6): 1422-1429.

LI Qi-feng, DING Jin-hua, DOU Wen-jing, ZHANG Hui, CHEN Chuang-fu. Effect on Expression of SUMO-1 in Cells Infected by Brucella and Construction of Lentivirus Vector that Over-expressing SUMO-1[J]. , 2016, 43(6): 1422-1429.

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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 Brucella and Construction of Lentivirus Vector that Over-expressing SUMO-1

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