Hepatitis B gene plant expression carrier plasmid, hepatitis B transgene cell line and its commercial production use
A plant cell and cell line technology, applied in the field of hepatitis B vaccine preparation, can solve the problems of not meeting social needs and low expression level
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Embodiment 1
[0040] Construction of Plant Cell Expression Vector of Hepatitis B Virus Surface Antigen Gene
[0041] 1. Extraction of chromosomal DNA from C28 cells
[0042] Take more than 80% of the adherent C28 cells (containing the HBV surface antigen S gene), wash the cells with PBS buffer, digest with 25% trypsin, resuspend the cells in TBS buffer, and harvest the cells by centrifugation. Resuspend the cells with TE (pH8.0) to make the concentration 5×107 / ml, take 1ml and add 10ml extraction buffer (10mmol / L Tris.Cl (pH8.0), 0.1mol / L EDTA (pH8.0) , 20ug / ml Trypsin, 0.5SDS), 37°C for 1 hour. Add proteinase K to a final concentration of 100ug / ml, and keep at 50°C for 3 hours. Cool to room temperature, add an equal volume of equilibrated phenol for extraction, and collect the aqueous phase by centrifugation. Add twice the volume of absolute ethanol, after mixing, centrifuge to discard the supernatant, wash the DNA precipitate with 75% ethanol, centrifuge to discard the supernatant, dry...
Embodiment 2
[0056] Expression vector transformed into Agrobacterium
[0057] 1. Preparation of Agrobacterium LBA4404 Competent Cells
[0058] Inoculate a single colony of Agrobacterium LBA4404 in 5ml YEP liquid medium, and culture overnight at 28°C and 220rpm. Take 2ml of the overnight culture and transfer it into 50ml of YEP liquid medium, culture at 28°C 220rpm until the OD600 is about 0.5, cool in ice for 30 minutes, centrifuge at 5000rpm at 4°C for 5 minutes, and discard the supernatant. Add 20ml of 50mmol / L CaCl2 to resuspend the bacteria, centrifuge at 5000rpm at 4°C for 5 minutes, and discard the supernatant. Add 2ml of 50mmol / L CaCl2 to resuspend the bacteria, aliquot 200ul per tube, and store at -80°C.
[0059] 2. Transformation of Agrobacterium
[0060] Take 20ng of the purified plasmid pBIBS, add it to 200ul competent Agrobacterium, mix well, ice bath for 5 minutes, transfer to liquid nitrogen to freeze for 8 minutes, and quickly incubate at 37°C for 5 minutes. Add 800ul YE...
Embodiment 3
[0064] Plant cell resistance sensitive pre-experiment
[0065] Ginseng callus cells were cultured in the following four mediums, and 1.5 g of ginseng callus solid culture cells were added to every 25 ml of medium.
[0066] 1. Ampicillin-resistant group: 60ul, 120ul, 180ul, 240ul, 360ul, 500ul of 50mg / ml ampicillin were added to 25ml 67V solid medium.
[0067] 2. Kanamycin-resistant group: 5ul, 10ul, 25ul, 50ul, 75ul, 100ul, 150ul of 50mg / ml kanamycin were added to 25ml 67V solid medium.
[0068] 3. G418 resistance group: 50 mg / ml G4185ul, 10ul, 25ul, 50ul, 75ul, 100ul, 150ul were added to 25ml 67V solid medium.
[0069] 4. Control group: 25ml 67V solid medium.
[0070] Comparing the growth situation within one month: there is no significant difference between the different resistance concentrations of the 4 groups and the 1 group, and the ginseng cells grow from 1.5g to 6~7g, indicating that the ginseng cells can grow normally in the presence of high-concentration ampicillin...
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