1.Correlation between Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes Signaling Pathway and Non-infectious Diseases.
Hua-Xiang YU ; Hai-Ying RUI ; Zi-Qi HAN ; Ruo-Chuan LI ; Lu-Lu LIU ; Li XUE ; Yu-Guo CHEN
Acta Academiae Medicinae Sinicae 2022;44(4):661-667
As a DNA receptor in the cytoplasm,cyclic GMP-AMP synthase (cGAS) can recognize abnormal DNA in the cytoplasm and activate stimulator of interferon genes (STING) to regulate the immune response. The recent studies have demonstrated that this pathway plays a role in non-infectious inflammatory diseases by promoting the expression of type Ⅰ interferon and interferon-stimulated gene.This article reviews the activation and regulation of cGAS-STING pathway in multiple systems and the effect of this pathway on the occurrence and progression of non-infectious inflammatory diseases,providing theoretical reference for future application of cGAS-STING pathway-related drugs in non-infectious inflammatory diseases.
Humans
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Interferons
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Membrane Proteins/metabolism*
;
Noncommunicable Diseases
;
Nucleotides, Cyclic
;
Nucleotidyltransferases/metabolism*
;
Signal Transduction
2.The E248R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immunity.
Yinguang LIU ; Wenping YANG ; Yuan WEN ; Qingli NIU ; Jifei YANG ; Guiquan GUAN ; Hong YIN ; Haixue ZHENG ; Dan LI ; Zhijie LIU
Chinese Journal of Biotechnology 2022;38(5):1837-1846
We researched the mechanism of African swine fever virus (ASFV) protein E248R in regulating the cGAS-STING pathway. First, we verified via the dual-luciferase reporter assay system that E248R protein inhibited the secretion of IFN-β induced by cGAS-STING or HT-DNA in a dose-dependent manner. The relative quantitative PCR analysis indicated that the overexpression of E248R inhibited HT-DNA-induced transcription of IFN-b1, RANTES, IL-6, and TNF-α in PK-15 cells. Next, we found that E248R interacted with STING by co-immunoprecipitation assay and laser confocal microscopy. Finally, we demonstrated that E248R inhibited the expression of STING protein by using Western blotting. We demonstrated for the first time that the E248R protein of ASFV suppressed the host innate immune response via inhibiting STING expression. The results are pivotal in extending the understanding of the ASFV immune escape and can guide the design of vaccines against ASFV.
African Swine Fever Virus/genetics*
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Animals
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DNA
;
Immunity, Innate
;
Nucleotidyltransferases/metabolism*
;
Signal Transduction
;
Swine
3.The I226R protein of African swine fever virus inhibits the cGAS-STING-mediated innate immune response.
Yabo LI ; Huicong LOU ; Yuna ZHAO ; Wenhui FAN ; Pengtao JIAO ; Lei SUN ; Tingrong LUO ; Wenjun LIU
Chinese Journal of Biotechnology 2023;39(12):4796-4808
This study aimed to explore the mechanism of how African swine fever virus (ASFV) I226R protein inhibits the cGAS-STING signaling pathway. We observed that I226R protein (pI226R) significantly inhibited the cGAS-STING-mediated type Ⅰ interferons and the interferon-stimulated genes production by dual-luciferase reporter assay system and real-time quantitative PCR. The results of co-immunoprecipitation assay and confocal microscopy showed that pI226R interacted with cGAS. Furthermore, pI226R promoted cGAS degradation through autophagy-lysosome pathway. Moreover, we found that pI226R decreased the binding of cGAS to E3 ligase tripartite motif protein 56 (TRIM56), resulting in the weakened monoubiquitination of cGAS, thus inhibiting the activation of cGAS and cGAS-STING signaling. In conclusion, ASFV pI226R suppresses the antiviral innate immune response by antagonizing cGAS, which contributes to an in-depth understanding of the immune escape mechanism of ASFV and provides a theoretical basis for the development of vaccines.
Animals
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Swine
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African Swine Fever Virus/metabolism*
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Membrane Proteins/metabolism*
;
Immunity, Innate
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Nucleotidyltransferases/metabolism*
;
Signal Transduction/genetics*
4.cGAS/STING signaling pathways induces the secretion of type Ⅰ interferon in porcine alveolar macrophages infected with porcine circovirus type 2.
Hongbo CHEN ; Feng LI ; Wenyan LAI ; Yuhao FANG ; Mingyong JIANG ; Dianning DUAN ; Xiaoyan YANG
Chinese Journal of Biotechnology 2021;37(9):3201-3210
In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals
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Cells, Cultured
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Circovirus
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Interferon Type I/genetics*
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Macrophages, Alveolar/virology*
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Membrane Proteins/metabolism*
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Nucleotidyltransferases/metabolism*
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Signal Transduction
;
Swine
5.Produce of marker-free transgenic tobacco plants by FLP/frt recombination system.
Xiao-Yi SHAN ; Bei LI ; Ju-Ren ZHANG
Chinese Journal of Biotechnology 2006;22(5):744-750
Selectable marker genes that usually encode antibiotic or herbicide resistances are widely used for the selection of the transgenic plants, but they become unnecessary and undesirable after transformation selection. An important strategy to improve the transgenic plants' biosafety is to eliminate the marker genes after successful selection. In the FLP/frt site-specific system of the 2 microm plasmid of Saccharomyces cerevisiae, the FLP enzyme efficiently catalyzes recombination between two directly repeated FLP recombination target (frt) sites, eliminating the sequence between them. By controlled expression of the FLP recombinase and specific allocation of the frt sites within transgenic constructs, the system can be applied to eliminate the marker genes after selection. Through a series of procedures, the plant FLP/frt site-specific recombination system was constructed, which included the frt containing vector pCAMBIA1300-betA-frt-als-frt and the FLP expression vector pCAMBIA1300-hsp-FLP-hpt. The FLP recombinase gene was introduced into transgenic (betA-frt-als-frt) tobacco plants by re-transformation. In re-transgenic plants, after heat shock treatment, the marker gene als flanked by two identical orientation frt sites could be excised by the inducible expression of FLP recombinase under the control of hsp promoter. Excision of the als gene was found in 41% re-transgenic tobacco plants, which indicated that this systerm could make a great contribution to obtain the marker free transgenic plants.
Base Sequence
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DNA Nucleotidyltransferases
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metabolism
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Molecular Sequence Data
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Plants, Genetically Modified
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genetics
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Polymerase Chain Reaction
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Promoter Regions, Genetic
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Recombination, Genetic
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Tobacco
;
genetics
6.Research progresses in synthetic biology of artemisinin.
Jian-Qiang KONG ; Wei WANG ; Ke-Di CHENG ; Ping ZHU
Acta Pharmaceutica Sinica 2013;48(2):193-205
Abstract: The first-line drug artemisinin is widely used against malaria. Commercially available artemisinin is extracted from plants. However, the lack of sufficient raw material, artemisinin and the cost associated with the drug's manufacture have limited the supply of ACT to most malaria sufferers in the Developing World. As such, it is important to develop a low cost, fine to environment and high-quality method to supply sufficient and reliable quantities of artemisinin in the future. The field of synthetic biology, which utilizes cell factories to manipulate microbial metabolism to enhance the production of artemisinin and its intermediates, has a particularly strong impact by providing new platforms for chemical production. After a brief introduction of the artemisinin biosynthetic pathway, the present review focuses on the introduction of artemisinin biosynthetic genes, such as the genes encoding amorpha-4, 11-diene monooxygenase, NADPH: cytochrome P450 oxidoreductase, artemisinic aldehyde delta 11(13) reductase and aldehyde dehydrogenase. The review also addresses general considerations for potential contributions of synthetic biology to artemisinin production, with an emphasis on factors influencing interest compounds production in chassis cells.
Antimalarials
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metabolism
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supply & distribution
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Artemisinins
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metabolism
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supply & distribution
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Biosynthetic Pathways
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Cytochrome P-450 Enzyme System
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genetics
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Escherichia coli
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metabolism
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Gene Dosage
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Genetic Engineering
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Isoenzymes
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genetics
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RNA Nucleotidyltransferases
;
genetics
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Retinal Dehydrogenase
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genetics
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Saccharomyces cerevisiae
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metabolism
;
Synthetic Biology
7.Cloning and expression analysis of 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase gene in Tripterygium wilfordii.
Yu-ru TONG ; Ping SU ; Meng ZHANG ; Yu-jun ZHAO ; Xiu-juan WANG ; Wei GAO ; Lu-qi HUANG
China Journal of Chinese Materia Medica 2015;40(22):4378-4383
To clone the 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (TwMCT) full length cDNA from Tripterygium wilfordii, the specific primers were designed according to the transcriptome data and the LCPCR were carried out. After a series of bioinformatics analysis on the TwMCT, the MeJA induced expression content were investigated by real-time fluorescence quantification polymerase chain reaction (RT-qPCR). The result showed that the full of TwMCTcDNA was 1 318 bp nucleotides encoding 311 amino acids. The molecular weight of the deduced TwMCT protein was about 34.14 kDa and the theoretical isoelectric point was 8.65. Result of the RT-qPCR analysis indicated that the content of TwMCT mRNA expression in T. wilfordii suspension cell was rising after treating with MeJA and reached the maximum in 24 h. Cloning and analyzing TwMCT gene from T. wilfordii provided gene element for studying the function and expression regulation of secondary metabolites.
Amino Acid Sequence
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Cloning, Molecular
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Erythritol
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analogs & derivatives
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metabolism
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Gene Expression Regulation, Plant
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Molecular Sequence Data
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Nucleotidyltransferases
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chemistry
;
genetics
;
metabolism
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Phylogeny
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Plant Proteins
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chemistry
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genetics
;
metabolism
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Protein Structure, Secondary
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Sequence Alignment
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Sugar Phosphates
;
metabolism
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Tripterygium
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chemistry
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enzymology
;
genetics
8.Prokaryotic expression of DNA recombinase FLP and its purification with enzymatic activity.
Ying GAI ; Wen-Qi WANG ; Hai LU ; Xiang-Ning JIANG
Chinese Journal of Biotechnology 2007;23(4):724-729
DNA recombinase FLP gene exists on the 2 micro plasmid of Saccharomyces cerevisiae. Recombinase FLP could recognize an FRT site composed of 34bp and function the sequences for exchange, recombination, deletion and reversion between the two orientated FRT sites. These functions are highly recognized by molecular biologists and biotechnology engineers for theoretic and applicable technology studies. This work constructed a prokaryotic over-expressed vector harboring FLP gene nominated as pQE30-flpe and established its over-expression culture system in which recombinase FLP could be efficiently expressed in E. coli strain M15. Purification procedures for high purity and active FLP are established through combination of ammonium sulfate precipitation with a 0.5-1.0 mL micro-column technique of Ni affinity chromatography with gradient elution. To verify the recombinase activity of purified FLP, substrate vectors, sequence donor vector (pUC18-FRT-gfp-FRT) and sequence accepting vector (pET30a-FRT) are constructed with various number, orientation of FRTs harboring the GFP gene for the expression of visible assay of the functions of recombination, exchange and deletion. Results showed that the system not only over expressed recombinase FLP in prokaryotic E. coli, but also efficiently purified the enzyme with a higher activity of the function of recombination, exchange and deletion. The system and the method are easily implemented and feasibly manipulated for theoretic study and biotechnology application.
Base Sequence
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DNA
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genetics
;
DNA Nucleotidyltransferases
;
biosynthesis
;
genetics
;
metabolism
;
Escherichia coli
;
genetics
;
metabolism
;
Fungal Proteins
;
metabolism
;
Molecular Sequence Data
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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isolation & purification
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Recombination, Genetic
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Saccharomyces cerevisiae
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enzymology
9.Structural insights into the assembly of human translesion polymerase complexes.
Wei XIE ; Xuan YANG ; Min XU ; Tao JIANG
Protein & Cell 2012;3(11):864-874
In addition to DNA repair pathways, cells utilize translesion DNA synthesis (TLS) to bypass DNA lesions during replication. During TLS, Y-family DNA polymerase (Polη, Polκ, Polı and Rev1) inserts specific nucleotide opposite preferred DNA lesions, and then Polζ consisting of two subunits, Rev3 and Rev7, carries out primer extension. Here, we report the complex structures of Rev3-Rev7-Rev1(CTD) and Rev3-Rev7-Rev1(CTD)-Polκ(RIR). These two structures demonstrate that Rev1(CTD) contains separate binding sites for Polκ and Rev7. Our BIAcore experiments provide additional support for the notion that the interaction between Rev3 and Rev7 increases the affinity of Rev7 and Rev1. We also verified through FRET experiment that Rev1, Rev3, Rev7 and Polκ form a stable quaternary complex in vivo, thereby suggesting an efficient switching mechanism where the "inserter" polymerase can be immediately replaced by an "extender" polymerase within the same quaternary complex.
Binding Sites
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Crystallography, X-Ray
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DNA Repair
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DNA-Binding Proteins
;
chemistry
;
genetics
;
metabolism
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DNA-Directed DNA Polymerase
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chemistry
;
genetics
;
metabolism
;
Fluorescence Resonance Energy Transfer
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Humans
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Mad2 Proteins
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Nuclear Proteins
;
chemistry
;
genetics
;
metabolism
;
Nucleotidyltransferases
;
chemistry
;
genetics
;
metabolism
;
Protein Binding
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Protein Structure, Quaternary
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Protein Structure, Tertiary
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Proteins
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chemistry
;
genetics
;
metabolism
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Recombinant Proteins
;
biosynthesis
;
chemistry
;
genetics
10.DNA sensor cGAS-mediated immune recognition.
Pengyan XIA ; Shuo WANG ; Pu GAO ; Guangxia GAO ; Zusen FAN
Protein & Cell 2016;7(11):777-791
The host takes use of pattern recognition receptors (PRRs) to defend against pathogen invasion or cellular damage. Among microorganism-associated molecular patterns detected by host PRRs, nucleic acids derived from bacteria or viruses are tightly supervised, providing a fundamental mechanism of host defense. Pathogenic DNAs are supposed to be detected by DNA sensors that induce the activation of NFκB or TBK1-IRF3 pathway. DNA sensor cGAS is widely expressed in innate immune cells and is a key sensor of invading DNAs in several cell types. cGAS binds to DNA, followed by a conformational change that allows the synthesis of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) from adenosine triphosphate and guanosine triphosphate. cGAMP is a strong activator of STING that can activate IRF3 and subsequent type I interferon production. Here we describe recent progresses in DNA sensors especially cGAS in the innate immune responses against pathogenic DNAs.
DNA, Bacterial
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immunology
;
metabolism
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DNA, Viral
;
immunology
;
metabolism
;
Gene Expression Regulation
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Host-Pathogen Interactions
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Humans
;
Immunity, Innate
;
Interferon Regulatory Factor-3
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genetics
;
immunology
;
Interferon Type I
;
biosynthesis
;
immunology
;
Membrane Proteins
;
genetics
;
immunology
;
Models, Molecular
;
NF-kappa B
;
genetics
;
immunology
;
Nucleotides, Cyclic
;
biosynthesis
;
immunology
;
Nucleotidyltransferases
;
genetics
;
immunology
;
Protein Binding
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Protein-Serine-Threonine Kinases
;
genetics
;
immunology
;
Signal Transduction