Method for producing recombinant human serum albumin by using human hepatoma carcinoma cell HepG2/C3A as bioreactor
A protein and cell membrane technology used in the field of recombinant human serum albumin production
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Embodiment 1
[0052] Example 1 Construction of vector PGK810 expressing AKT1 gene (myriAKT1) containing SRC protein membrane localization signal and activated MEK1S2E gene
[0053] myriAKT1 and MEK1SS2EE genes are linked by IRES (myriAKT1-IRES-MEK1SS2EE, see figure 1 ). The AKT1 gene (SEQ ID NO: 3) containing the coding sequence and amino acid sequence (SEQ ID NO: 1, 2) of the SRC protein myristylation peptide at the 5' end was fully synthesized, and the amino acid sequence obtained by expression is shown in SEQ ID NO: 4. Totally synthetic IRES fragment (SEQ ID NO: 5). The MEK1SS2EE gene (SEQ ID NO: 6) and the amino acid sequence of MEK1SS2EE (SEQ ID NO: 7) were synthesized. Using the vector part of PGK-H2BmCherry (Addgene Plasmid #21217) digested with restriction endonucleases BamHI and SalI as the backbone, myriAKT1, IRES and MEK1SS2EE were spliced and connected to the BamHI of the PGK-H2BmCherry vector by multi-fragment recombination splicing and SalI site, the vector PGK810 was obt...
Embodiment 2
[0054] Embodiment 2 prepares PGK810 virus
[0055] Taking 1 well in a six-well plate as an example, each sample needs 1.3×10 6 293T cells.
[0056] 1. On the first day, plant 1.3×10 cells in 6-Well cell culture wells 6 293T cells, the cell culture medium is 2mlDMEM(GIBCO)+10%FBS(GIBCO). Set at 37°C, 5% CO 2 Incubator for 16 hours.
[0057] 2. On the second day, take a 1.5ml sterilized EP tube, add 1.6μg PGK810 plasmid, 1.2μg packaging auxiliary plasmid PAX2 and 0.8μg packaging auxiliary plasmid VSV-G, add 200μl opti-MEM, let stand for 5 minutes, then add 10.8 μl liposome transfection reagent Fugene HD (Roche), let stand for 15 minutes.
[0058] 3. Evenly add opti-MEM containing DNA and Fugene HD liposomes to 293T cells in 6-Well, mix slightly, and return the cells to 37°C, 5% CO 2 The cell culture incubator was continued for 12 hours.
[0059] 4. 12 hours after transfection, replace the medium in the 6-Well with 2ml fresh DMEM+10%FBS fully preheated, and place at 37°C, ...
Embodiment 3
[0061] Example 3 PGK810 virus infection of C3A cells and serum-free screening
[0062] 1, 1.3×10 6 C3A cells (ATCC Number: CRL-10741) were planted in 10cm cell culture dish with 10ml DMEM / F12 medium containing 10%FBS, 37℃, 5%CO 2 The incubator cultivated for 12hr.
[0063] 2. Add 12μl 8mg / ml polybrene to the petri dish and mix well, add 2ml of PGK810 virus, continue culturing for 12hrs and replace with fresh medium.
[0064] 3. After continuing to cultivate for 36 hours, replace the original medium with 10ml of fresh medium, and continue to cultivate for 24 hours.
[0065] 4. Digest the cells with trypsin, inoculate them in fresh medium and a new culture dish at a ratio of 1:5, after 24 hours, replace with serum-free DMEM / F12 medium, and continue to culture for 72 hours.
[0066] 5. Collect all suspended cells, digest unsuspended adherent cells with trypsin, combine all cells, inoculate in fresh serum-free DMEM / F12 medium at a ratio of 1:5, and continue to culture for 24 ho...
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