Screening and detection of repetitive extragenic palindromic sequences with TLR9 agonistic activity from Brucella abortus DNA
ZHANG Ya-xian1,2, BAI Li-yun2, WANG Zhan-li2, WANG Ying2, YU Hui3
1. Baotou Medical College, Baotou 014040, China; 2. The First Affiliated Hospital of Baotou Medical College, Baotou 014040, China; 3. The Second Affiliated Hospital of Baotou Medical College, Baotou 014040, China; 4.Chifeng City for Disease Control and Prevention,Chifeng 024000,China
Abstract:The aim of this study was to screen natural DNA sequences named repetitive extragenic palindromic sequences (REPs) with TLR9 agonistic activity from Brucella abortus DNA. Complete genome sequence of Brucella abortus A13334 was available in GenBank. A total of 1 857 REP sequences in Brucella abortus was identified. Bioinformatics technology was used to identify Brucella abortus REPs. Five REP sequences were synthesized for further detecting the TLR9 agonistic activity. Phosphodiester ODNs representing the selected ODNs were then synthesized. Murine macrophage RAW264.7 were cultured and transfected with ODNs mediated by lipofectamine 3000. The content of IFN-α in the supernatant was then measured by ELISA. We found that IFN-α expression was significantly increased in RAW264.7 treated with M4 and M5. In addition, TLR9-siRNA was used to down regulate TLR9 expression. IFN-α levels were examined in RAW264.7 transfected with TLR9-siRNA upon ODN stimulation. Results showed TLR9-siRNA could significantly reduce the levels of IFN-α in RAW264.7 treated with M5. This work demonstrated that REP sequences present in Brucella abortus DNA were able to produce macrophages stimulation through TLR9.
张雅娴, 白丽云, 王占黎, 王英, 于慧. 活化TLR9的牛种布氏菌基因外重复回文序列筛选及活性检测[J]. 中国人兽共患病学报, 2016, 32(5): 447-450.
ZHANG Ya-xian, BAI Li-yun, WANG Zhan-li, WANG Ying, YU Hui. Screening and detection of repetitive extragenic palindromic sequences with TLR9 agonistic activity from Brucella abortus DNA. Chinese Journal of Zoonoses, 2016, 32(5): 447-450.
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