Coding

Part:BBa_K2913019

Designed by: Yuhan Liu, Yannan Wang   Group: iGEM19_NEFU_China   (2019-10-20)


TNF-α

Tumor necrosis factor (TNF, cachexin, or cachectin; once named as tumor necrosis factor alpha or TNFα) is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.TNF is a member of the TNF superfamily, consisting of various transmembrane proteins with a homologous TNF domain. The primary role of TNF is in the regulation of immune cells. TNF, being an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumorigenesis and viral replication and respond to sepsis via IL1- & IL6-producing cells. Dysregulation of TNF production has been implicated in a variety of human diseases including Alzheimer's disease, cancer, major depression, psoriasis and inflammatory bowel disease (IBD). Though controversial, studies of depression and IBD are currently being linked to increased levels of TNF. Recombinant TNF is used as an immunostimulant under the INN tasonermin. TNF can be produced ectopically in the setting of malignancy and parallels parathyroid hormone both in causing secondary hypercalcemia and in the cancers with which excessive production is associated.


Usage and Biology

result

As we can see that the TNF-α is expressed after the induction, and TNF-α sample for 50% volume can kill 82.5% HepG2 cells, the TNF-α sample for 20% can kill 70.8% HeLa cells. Eventually, our system is able to induced by hypoxia and high lactic acid to secrete cytotoxic TNF-α, causing the evident death of HeLa and HepG2 cells.

Fig. OD450 value can represent the cell number. “TNF-α” is the very group containing TNF-α theoretically. (a) Different volume of the supernatant treated the HeLa cells for 24h. (b) Different volume of the supernatant treated the HepG2 cells for 24h.

As we can see that the TNF-α is expressed after the induction, and TNF-α sample for 50% volume can kill 82.5% HepG2 cells, the TNF-α sample for 20% can kill 70.8% HeLa cells. Eventually, our system is able to induced by hypoxia and high lactic acid to secrete cytotoxic TNF-α, causing the evident death of HeLa and HepG2 cells.

MIT_MAHE 2020

Summary

Tumor necrosis factor is a cell signaling protein (cytokine) involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activated macrophages, although it can be produced by many other cell types such as CD4+ lymphocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons. The primary role of TNF is in the regulation of immune cells. TNF, being an endogenous pyrogen, is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumorigenesis and viral replication and respond to sepsis via IL1- & IL6-producing cells. Dysregulation of TNF production has been implicated in a variety of human diseases including Alzheimer's disease, cancer, major depression, psoriasis and inflammatory bowel disease (IBD).

References

[1] Old, L.J., Tumor necrosis factor (TNF). Science, 1985. 230(4726): p. 630-2.

[2] Watanabe, N., et al., Toxic effect of tumor necrosis factor on tumor vasculature in mice. Cancer Res, 1988. 48(8): p. 2179-83.

[3] Folli, S., et al., Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability. Int J Cancer, 1993. 53(5): p. 829-36.



Contribution from Squirrel-Beijing 2024

Expression detection of the special functional protein and the related inflammatory pathway by western blot: The treated cells in Part 1 were collected and treated with lysis buffer, 300 μL of the 5× loadding buffer was added into each cell lysis sample, and we boiled and denatured the samples for 15 min. To quantify protein expressions of PLP-1, associated proteins PGC-1α and TNF-α, with respect to the control protein β-actin, western blot experiment was performed. 1× Tris-glycine electrophoresis buffer was filled into the electrophoresis unit, samples and pre-stained rainbow protein marker were loaded onto the gel plates and electrophoresed initially at 120V for 15 min, then at 120V for about 40 min until the protein marker was completely separated. The PVDF membranes were prepared by one-minute immersion in anhydrous methanol. The membranes, sponges, and filter paper were submerged in a 1× transfer buffer, and the assembly sequences within the transfer apparatus was organized from the negative to positive pole as follows: sponge, filter paper, gel, membrane, filter paper, and sponge, and the membranes then underwent a 110V transfer for 1.5 h. After transfer, the membranes were rinsed and incubated in 10% skimmed milk for an hour. The membranes were then washed using 1× TBST with 5 min for three times, which were further incubated with the primary antibodies for two hours. Later, the membranes were washed using 1× TBST buffer for three times, and next incubated with goat anti-rabbit IgG-HRP and 1× TBST for 1 h. After three times of 1× TBST washing, 1 mL ECL developer for the membrane was prepared in darkness and added to the membrane, and chemiluminescence signals were captured with a designated imaging system, and the relative amounts of proteins in each group were quantified concerning β-actin expression. Results demonstrated that the combinations of Vb6 and Vb12 with the ratio of 10:1 and 20:1 had the strongest anti-inflammatory effects, embodying on the up-regulation of PLP-1 and PGC-1α proteins, as well as the down-regulation of TNF-α protein.

图1

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1220
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1220
    Illegal NheI site found at 250
    Illegal NheI site found at 273
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1220
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1220
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1220
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 85
    Illegal BsaI site found at 1310


[edit]
Categories
//collections/immune_regulation/inflammatory
Parameters
None