Interleukin-2-Huaqiang electronic network

L06ESDL5V0H4-2

C-2

Other names: T cell growth factor (TCF)

definition:

A broadly biologically active cytokine produced primarily by activated CD4+ T cells and CD8+ T cells. It is a growth factor for all T cell subsets and promotes the proliferation of activated B cells. Therefore, it is an important factor regulating immune response and is also involved in antibody response, hematopoiesis and tumor surveillance.

[Chinese name]: Interleukin-2

[Short Pinyin]: BXBJS-2

[English name]: Interleukin-2

[Category]:Immuno-enhancement agent||Other anti-tumor drugs and adjuvant medicines

[alias] leucovorin, hinjier, T cell growth factor, interleukin-2

[foreign name] Interleukin-2, IL-2

[Indications] Interleukin-2 is a glycoprotein with a molecular weight of 14,500 that stimulates the proliferation of T cells that have been activated by specific antigens or mitogenic factors. Recombinant interleukin-2 can be used in clinical research. Mainly used for kidney cancer, melanoma and non-Hodgkin's lymphoma (Rossenberg). LAK cells represent a heterogeneous cell body characterized by mononuclear macrophages, B and T lymphocytes. These LAK cells appear to have a specific effect on cancer cells. Furthermore, LAK cells (unlike T cells) mediate killing without being bound by MHC. [1]

1 Materials and methods:

1.1

Prepare crude IL-2 aseptic blood collection, heparin anticoagulation, dilute the blood with RPMI1640 1:1, then re-layer on phenanthrene solution, centrifuge at 1500r/min for 30min, take leukocyte layer, wash twice with RPMI1640 Add 10% fetal bovine serum RPMI1640 at a concentration of 1~3×10 cells/ml, inject into a large bottle equipped with a magnetic rod, stir and incubate at 37 °C for 4 days, take out, and dispense in a 50 ml centrifuge tube, 1500 r/min. After centrifugation for 20 min, the supernatant was discarded, and the cells were suspended in 1% fetal bovine serum RPMI1640 medium to give a cell concentration of 10

6

Cell/ml. At the same time, pure PHA1～2μg/ml and various B lymphoblastoid cells were added to make the concentration 10

6

The cells/ml were stirred and cultured at 37 ° C for 18-24 hours, and then centrifuged at 1500 r/min for 30 min. The supernatant was taken and sterilized by filtration through a 045 μg filter. The filtrate was crude IL-2.

2 Preparation of fine IL2

2.1 Ammonium sulfate precipitation and vacuum concentration dialysis:

The ammonium sulfate powder was added to the above crude IL-2 at a concentration of 50% at 4 ° C, stirred for 2 h, centrifuged at 12000 g for 30 min, and the supernatant was taken, and then 80% saturated ammonium sulfate was added thereto, overnight at 4 ° C, and the next day was 12000 g. After centrifugation for 30 min, the supernatant was discarded, and the precipitate was dissolved in 1% PEG6000 in a 1:2 PBS solution (pH 73), and concentrated in a vacuum negative pressure dialyzer to a desired volume.

2.2 Sephadex G100 gel filtration:

Filter through a 2 x 180 cm filter column and PBS (ibid.) at a flow rate of 80 ml/h. Each tube eluate was measured for its OD value by an ultraviolet spectrophotometer at 280 nm. At the same time, several standard proteins with different molecular weights were also filtered and tested for OD values ​​under the same conditions, and the curves of IL-2 and standard protein OD values ​​were plotted. After the tube eluate was filtered and sterilized, the leukocyte biological activity test was performed, and the curve was drawn according to the test results, and several tube eluates having the highest IL2 activity were found, and after mixing, the next step was refined.

2.3 BlueSepharose chromatography: The above mixed eluate was added to a BlueSepharose column at a flow rate of about 15 ml/h. The effluent was collected in 10 ml per tube. After all the samples flowed out of the column, the sample column was washed with the above PBS, and then the gradient was used. The NaCl solution (from 0075 mol/L to 06 mol/L NaCl in PBS) was eluted, and the eluate was collected in 2 ml/tube. The gradient and flow rate of the NaCl solution were all controlled by a gradient mixer. After the elution is completed, the OD value and the NaCl concentration of each tube are measured. After the tube eluate was filtered and sterilized, the biological activity was measured. Mixing the tube eluates before and after the peak is the refined IL2. Store in a -20 ° C refrigerator.

3 verification

3.1 Activity determination The above IL2 was diluted to 50%, 25%, 125%, and 625% with 10% fetal bovine serum RPMI1640, and then each percentage concentration and 100% concentration of IL2 were diluted by a ratio of 1:2 to At 1:128, each dilution was separately added to the wells of the multiwell plate at 01 ml per well. Then, CT6 cell line (IL2-dependent T cell strain of mouse) was taken, adjusted to a concentration of 106 cells/ml, and 01 ml was added to each well, and medium was used instead of IL2 as a control. After the perforated plate was cultured for 24 hours, it was taken out and the H3-labeled thymidine was added at a concentration of 1 μci per well for 4 hours. After the removal, the cells were washed with a MESHII cell harvester, and the cpm of each well was measured with a beta counter. If necessary, plot the curve according to this result to determine the activity unit.

3.2 Determination of protein content Using spectroscopy, OD280 and OD260 were measured, respectively, and then calculated as follows: protein content (mg / ml) = (144 × OD280-075 × OD260) × dilution factor

3.3 Toxicity test and bacterial test refer to the preparation part of thymosin.

application

[Pharmacological action] This product can promote the proliferation and differentiation of T lymphocytes, induce killer cells with cytotoxic activity, induce and enhance the viability of killer T cells, monocytes and macrophages.

[Clinical application] for chronic active hepatitis B virus infection, etc.

[Usage and Dosage] Intravenous drip: 25,000-50000 U each time, once a day.

[Adverse reactions] Common reactions such as fever and chills. 1. Digestive system: nausea, vomiting, diarrhea, local necrosis of the colon or perforation. 2. Respiratory system: can cause interstitial pulmonary edema, respiratory alkalosis. 3. Nervous system: behavioral changes and cognitive impairments can occur. 4. Cardiovascular system: symptoms such as hypotension, tachycardia, and arrhythmia may occur. 5. Blood system: neutrophils can rise, lymph and monocytes decrease. 6. Urinary system: oliguria, fluid retention, and ammoniaemia. Endocrine dysfunction can occur, blood calcium, blood phosphorus and vitamin C deficiency.

[Specifications] powder injection: 1000u, 10000u, 100,000 u, 1 million u.

Note: epilepsy, severe hypotension, heart and kidney dysfunction, high fever disabled. Pregnant women, patients with heart disease or lung disease, and those over 60 years old should be used with caution. Liver and kidney function should be checked regularly during medication.