Preparation and verification of interleukin 2

Preparation and verification of interleukin 2

(1) Principle Interleukin 2 (IL2) is a soluble glycoprotein produced by Th cells stimulated by mitogens or specific antigens and with the help of interleukin 1. IL2 is an important broad-spectrum immune enhancing factor in the body, and is commonly used in clinical treatment of immunodeficiency diseases and tumors.

(2) Materials and methods

1 Prepare crude IL2 for aseptic blood collection, heparin anticoagulation, dilute the blood with RPMI1640 at 1: 1, and then re-layer on the Ficoll liquid surface, centrifuge at 1500r / min for 30min, take the leukocyte layer, wash twice with RPMI1640 ， Add 10% fetal bovine serum RPMI1640 at a concentration of 1 ～ 3 × 106 cells / ml, inject it into a large bottle with a magnetic rod, incubate at 37 ℃ for 4 days, take it out, and distribute it in a 50ml centrifuge tube, 1 500r / Centrifuge for 20 minutes at min, discard the supernatant, and suspend the cells in 1% fetal bovine serum RPMI1640 medium to make the cell concentration 106 cells / ml. Simultaneously add pure PHA 1-2μg / ml and a variety of B lymphoblastoid cell strains to a concentration of 106 cells / ml. After stirring and culturing at 37 ℃ for 18-24 hours, take out and centrifuge at 1500r / min for 30min. Take the supernatant and use 045μg filter membrane was sterilized by filtration, and the filtrate was crude IL2.

2 Preparation of fine IL2

(1) Ammonium sulfate precipitation and vacuum concentration dialysis: add ammonium sulfate powder to the above crude IL2 at 50% saturation at 4 ° C, stir for 2h, centrifuge at 12,000g for 30min, take the supernatant, then add 80% saturated sulfuric acid Ammonium, overnight at 4 ° C, centrifuge at 12 000g for 30 min the next day, discard the supernatant, dissolve the precipitate in a 1: 2 PBS solution (pH 73) of 01% PEG 6 000, and place it in a vacuum negative pressure concentrating dialyzer for dialysis Concentrate to the required capacity.

(2) Sephadex G100 gel filtration: filter with a 2 × 180 cm filter column and PBS (same as above) at a flow rate of 80 ml / h. The eluate of each tube was tested for its OD value with an ultraviolet spectrophotometer at 280 nm. At the same time, several standard proteins with different molecular weights were also filtered and their OD values ​​were detected under the same conditions, and the curves of IL2 and standard protein OD values ​​were plotted. After filtering and sterilizing the eluate of each tube, a leukocyte biological activity test is performed, and a curve is drawn according to the test result, and a few tubes of eluent with the highest IL2 activity are found.

(3) BlueSepharose chromatography: add the mixed eluate to the BlueSepharose column at a flow rate of about 15ml / h, collect the effluent according to 10ml per tube. After all the sample flows out of the column, wash the sample column with the above PBS, then Elute with a gradient NaCl solution (from 0075 mol / L to 06 mol / L NaCl in PBS) and collect the eluate in 2 ml / tube. The gradient and flow rate of the NaCl solution are regulated and controlled by the gradient mixer. After the elution, the OD value and NaCl concentration of each tube were measured. After filtering and sterilizing the eluate of each tube, biological activity was determined. The elution solution of several tubes before and after the peak is mixed, which is the purified IL2. Store in -20 ℃ refrigerator.

(3) Verification

1 For activity determination, dilute the above IL2 to 50%, 25%, 125%, 625% with 10% fetal bovine serum RPMI1640, and then dilute each percentage and 100% concentration of IL2, from 1: 2 to 1: 128, add each dilution to the wells of the multi-well plate, 01ml per well. Then take the CT6 cell line (the mouse IL2-dependent T cell line), adjust the concentration to 106 cells / ml, add 01 ml per well, and use the medium instead of IL2 as a control. After culturing the multi-well plate for 24h, take out the thymidine labeled with thymidine at the concentration of 1μci per well and continue to culture for 4h. After taking out, the cells were filtered and washed with a MESH II cell collector, and the cpm of the cells in each well was measured with a β counter. If necessary, draw a curve based on this result to determine the unit of activity.

2 Determination of protein content using spectrophotometer, respectively measured OD280 and OD260, and then calculated according to the following formula: protein content (mg / ml) = (144 × OD280-075 × OD260) × dilution factor

3 Refer to the preparation of thymosin for toxicity test and bacterial inspection.

(Four) matters needing attention

The preparation process of IL2 is similar to the process of cell culture, so its precautions are also similar to cell culture. The measurement of IL2 activity uses the isotope incorporation method, so it must be operated as required to prevent isotope diffusion and contamination.