Application of HepG2.2.15 cell autophagy corpuscle in preparing hepatitis b virus (HBV) therapeutic vaccines
A therapeutic vaccine and autophagosome technology, applied in animal cells, tumor/cancer cells, antibody medical components, etc., can solve the problems of no therapeutic vaccine approved for marketing and unstable overall curative effect
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Embodiment 1
[0032] Example 1: Preparation of autophagosome HBV-DRibbles in HepG2.2.15 cells.
[0033] 1. Culture of HepG2.2.15 liver cancer cells:
[0034] To recover the frozen cells, use DMEM medium containing 10% (v / v) fetal bovine serum, 100 U / ml penicillin and 100 U / ml streptomycin at 37°C, 5% (v / v) CO 2 Cultured in a constant temperature incubator, the medium was changed every 1 to 2 days, and routinely digested and passaged with 0.25% trypsin.
[0035] 2. Treatment of HepG2.2.15 liver cancer cells:
[0036] After the cells were well attached to the wall and the density was moderate, a combination of rapamycin 100nmol / L, Velcade 200nmol / L, and ammonium chloride 30mmol / L was added to intervene HepG2.2.15 cells for 16 hours to induce autophagosomes HBV-DRibbles.
[0037] 3. Extract HBV-DRibbles:
[0038] After the cells were treated with drugs for 16 hours, the cells were harvested, and the DRibbles in the supernatant were extracted by centrifugation. Specific steps are as follows...
Embodiment 2
[0044] Example 2: Identification of the morphology of HBV-DRibbles by transmission electron microscopy.
[0045] The extracted HBV-DRibbles were centrifuged at high speed to compress them tightly, the supernatant was discarded, the precipitate was fixed with 2.5% (v / v) glutaraldehyde, and sent to the electron microscope room for processing, and the morphology of HBV-DRibbles was observed by the microscope. Such as figure 1 Shown: Under the electron microscope, the body with membrane structure can be seen, with an average diameter of about 200-300nm (pointed by the arrow), which proves that the autophagosome of liver cancer cells is effectively recruited.
Embodiment 3
[0046] Example 3: Determination of total protein content of HBV-DRibbles (Bradford method).
[0047] 1) Completely dissolve the protein standard, take 25 μl and dilute to 100 μl with PBS;
[0048] 2) Add 0, 1, 2, 4, 8, 12, 16, and 20 μl of the standard into the 96-well plate, and add PBS to make up to 20 μl;
[0049] 3) Freeze and thaw the DRibbles to be tested repeatedly in advance, centrifuge to take the supernatant, add an appropriate volume of samples to a 96-well plate, and add PBS to make up to 20 μl;
[0050] 4) Add 200 μl of G250 staining solution to each well and let stand at room temperature for 3-5 minutes;
[0051] 5) Measure the absorbance at 595nm or other wavelengths between 560-610nm with a microplate reader;
[0052] 6) Draw a standard curve with the software ElisaCalc, and calculate the protein concentration in the sample according to the standard curve.
[0053] The protein concentration of each extraction is not exactly the same, about 1-3μg / μl.
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