Difference between revisions of "Part:BBa K2986004"

Revision as of 08:26, 7 October 2019

hGluc

Usage and Biology

The luciferase from the marine copepod Gaussia princeps (Gluc) is the smallest (∼19.9-kDa) coelenterazine (CTZ)-utilizing luciferase known thus far. A humanized variant of Gluc (hGluc), which was codon optimized for expression in cultured mammalian cells, has been widely used as a reporter gene. Gaussia luciferase exhibits an activity up to 1,000-fold higher than to Renilla reniformis luciferase (Rluc), firefly luciferase (Fluc), or bacterial luciferases (LuxAB). The outstanding sensitivity of Gluc-based assays was previously demonstrated detecting as low as 10−18 mol purified Gluc or one eukaryotic cell transiently expressing Gluc.

Figjre1.the plasmid used with hgluc

we use the plamid contains the hGluc, mRuby to be a quantity that characterize the the level of secretion the cytokine Il8. Interleukin 8 (IL8 or chemokine (C-X-C motif) ligand 8, CXCL8) is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells and endothelial cells. Endothelial cells store IL-8 in their storage vesicles, the Weibel-Palade bodies. In humans, the interleukin-8 protein is encoded by the CXCL8 gene. IL-8 is initially produced as a precursor peptide of 99 amino acids which then undergoes cleavage to create several active IL-8 isoforms.In culture, a 72 amino acid peptide is the major form secreted by macrophages.

There are many receptors on the surface membrane capable of binding IL-8; the most frequently studied types are the G protein-coupled serpentine receptors CXCR1 and CXCR2. Expression and affinity for IL-8 differs between the two receptors (CXCR1 > CXCR2). Through a chain of biochemical reactions, IL-8 is secreted and is an important mediator of the immune reaction in the innate immune system response.

IL-8 can be secreted by any cells with toll-like receptors that are involved in the innate immune response. Usually, it is the macrophages that see an antigen first, and thus are the first cells to release IL-8 to recruit other cells. Both monomer and homodimer forms of IL-8 have been reported to be potent inducers of the chemokine receptors CXCR1 and CXCR2. The homodimer is more potent, but methylation of Leu25 can block the activity of homodimers.

IL-10 is a cytokine with multiple, pleiotropic, effects in immunoregulation and inflammation. It downregulates the expression of Th1 cytokines, MHC class II antigens, and co-stimulatory molecules on macrophages. It also enhances B cell survival, proliferation, and antibody production. IL-10 can block NF-κB activity, and is involved in the regulation of the JAK-STAT signaling pathway.

Discovered in 1991 IL-10 was initially reported to suppress cytokine secretion, antigen presentation and CD4+ T cell activation. Further investigation has shown that IL-10 predominantly inhibits lipopolysaccharide (LPS) and bacterial product mediated induction of the pro-inflammatory cytokines TNFα, IL-1β, IL-12, and IFNγ secretion from Toll-Like Receptor (TLR) triggered myeloid lineage cells.

Experiment

Figjre2.hGluc flow and Il8 flow

Figjre3.hGluc secretion and Il8 Elisa

</p> We use the hGluc to be a quantity that characterize the the level of secretion the cytokine Interleukin 8. We can see from the curve that the tendency of the hGluc is similiar to the secertion of the Il8. This shows our plasmid works will in transfering the hGluc-bound cytokine expression system into the target cells.

Figjre4.IL 10 (102.4uw light intensity) data fitting & parameter estimation

we set up a mathematic model to stimnulate the process of expressing the cytokine and Notation: X: monomer, X*: dimer, D: promoter, P: polymerase, 𝑌_𝑚: mRNA. Parameter: 𝑐_1: total # of X, 𝑐_2: total # of D, c: light density, a: top, b: bottom, 𝑛_1&𝑛_2: hill slope, 𝐾_1&𝐾_2: activation coefficient.

Figjre5.The mathematic model

Design Notes

how to use the hGluc to represent the cytokine expression.

References

[1].Tannous B. A. (2009). Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nature protocols, 4(4), 582–591. doi:10.1038/nprot.2009.28.

[2].Hedges JC, Singer CA, Gerthoffer WT (2000). "Mitogen-activated protein kinases regulate cytokine gene expression in human airway myocytes". Am. J. Respir. Cell Mol. Biol. 23 (1): 86–94. CiteSeerX 10.1.1.326.6212. doi:10.1165/ajrcmb.23.1.4014. PMID 10873157.

[3].Brat DJ, Bellail AC, Van Meir EG (2005). "The role of interleukin-8 and its receptors in gliomagenesis and tumoral angiogenesis". Neuro-oncology. 7 (2): 122–133. doi:10.1215/s1152851704001061. PMC 1871893. PMID 15831231.

[4].Utgaard JO, Jahnsen FL, Bakka A, Brandtzaeg P, Haraldsen G (1998). "Rapid secretion of prestored interleukin 8 from Weibel-Palade bodies of microvascular endothelial cells". J. Exp. Med. 188 (9): 1751–6. doi:10.1084/jem.188.9.1751. PMC 2212514. PMID 9802986.

[5].Modi WS, Dean M, Seuanez HN, Mukaida N, Matsushima K, O'Brien SJ (1990). "Monocyte-derived neutrophil chemotactic factor (MDNCF/IL-8) resides in a gene cluster along with several other members of the platelet factor 4 gene superfamily". Hum. Genet. 84 (2): 185–7. doi:10.1007/BF00208938. PMID 1967588.

Sequence and Features