Analysis of genes induced by bacterial immunostimulatory CpG-DNA

细菌DNA和免疫刺激寡核苷酸(ISS-ODN)可诱导巨噬细胞、单核细胞和树突状细胞分泌IL-6、IL-12和TNF-α等促炎细胞因子，并上调MHC-II、CD40和CD86的表达。为了进一步研究细菌DNA中特定模式识别的分子后果，我们使用了一种名为抑制消减杂交(SSH)的敏感的基于PCR的消减方法来分离ISS-ODN刺激的小鼠脾细胞中上调的基因。通过SSH分离的87个克隆的序列分析表明，41个克隆与BLAST程序中已知的小鼠蛋白完全一致。6个克隆与已知的人或大鼠蛋白有80%-90%的同源性。有31个克隆与已知的未分配任何功能的基因产物同源。其余9个克隆与公共数据库中的任何序列都没有显著的同源性。RT-PCR证实了在ISS-ODN刺激的细胞中过表达的核因子-kB p105、干扰素调节因子-1(IRF-1)、蛋白酶体激活物(PA)28b、IRG2(干扰素-IP)和MyD88。

Bacterial DNA and immunostimulatory oligodeoxyribonuceotides (ISS-ODN) can induce macrophages, monocytes, and dendritic cells to secrete proinflammatory cytokines such as IL-6, IL-12 and TNF-a, and up-regulate MHC class II, CD40 and CD86 expressions. To further characterize molecular consequences of the recognition of a specific pattern in bacterial DNA, we used a sensitive PCR-based subtraction approach, termed suppression subtractive hybridization (SSH), to isolate genes upregulated in ISS-ODN-stimulated murine spleen cells.Sequence analysis of 87 clones isolated by SSH revealed that 41 clones were found to correspond exactly to known mouse protein in the BLAST program. Six clones were revealed to have 80-90% homology with known human or rat proteins. Thirty-one clones showed homology with known cDNA whose gene products are not allocated to any functions. Remaining 9 clones showed no significant homology with any sequences in the public database.Overexpression of NF-kB p105, IFN regulatory factor-1 (IRF-1), proteasome activator (PA) 28b, IRG2 (IFN-IP), and MyD88, which have not previously been reported as overexpressed in the ISS-ODN-stimulated cells, were confirmed by using RT-PCR.