A kind of cell line for protein display and expression, preparation method and application thereof
A protein display and cell technology, applied in the field of protein display cell lines and their preparation, can solve the problems of affecting RNA polymerase II transcription activity, complex chromosome structure, difficulties and the like
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Embodiment 1
[0029] Example 1. Materials and Methods
[0030] 1. Cell culture
[0031] CHO-K1 cells and a series of cells constructed with CHO-K1 were used PRMI-1640 medium containing 10% fetal bovine serum (Hyclone) and 100U / mL double antibody at 37°C, 5% CO. 2 Cultivated in an incubator. CHO / dhFr-(dihydrofolate reductase-deficient Chinese hamster ovary cells) were grown in IMDM medium containing 10% fetal bovine serum (Hyclone), 100 U / mL double antibody, 0.1 mM hypoxanthine and 0.016 mM thymine. 37°C, 5% CO 2 Cultivated in an incubator.
[0032] Human 293F cells using FreeStyle TM 293Expression Medium at 37°C, 5% CO 2 Incubator, shake flasks at 120 rpm.
[0033] 2. Cell transfection and establishment of cell lines
[0034] In order to build a stable cell line integrated at a specific site, CHO-K1 cells were seeded into 6-well plates one day in advance, and the next day, when the cells were expanded to 60%-80% of their full capacity, CHO-K1 cells were mixed with 500ng of donor plas...
Embodiment 2
[0045] Example 2. Construction of CHO cell line for protein display
[0046] In the present invention, a replaceable expression cassette is inserted into a specific site in the CHO cell genome, thereby constructing a high-expressing cell line of a single-copy gene. This cell line can rapidly replace any antibody gene or other genes into the genome, achieve high transcription and expression of antibody genes, and display the antibody on the surface of CHO cell membrane for affinity maturation of the antibody.
[0047] In the present invention, the YWHAE gene in the CHO cell genome is selected as the site-directed insertion position of the replaceable expression cassette, and three sites of the YWHAE gene are selected for site-directed insertion of the replaceable expression cassette. These three sites are E site (exon1), I site (intron, between exon 3 and exon 4) and T site (behind the YWHAE gene stop codon), as shown in figure 1 shown.
[0048] The inserted replaceable express...
Embodiment 3
[0052] Embodiment 3, double histase-mediated target gene in-frame replacement
[0053] In order to test whether the obtained CHO-K1-T cell line can carry out double histone-mediated frame replacement and the corresponding protein expression (in the following embodiments, the CHO-K1-T cell line can also be modified with T31 replace). We replaced the GFP gene in the clone with RFP gene, anti-TNF-αScFv antibody gene and anti-HMGB1 ScFv antibody gene by RMCE. Cells expressing RFP or displaying antibodies were then sorted by flow cytometry. Sorted cells were expanded in culture and collected for flow analysis, with representative results such as figure 2 shown.
[0054] like figure 2 As shown, the GFP gene in CHO-K1-T can be successfully replaced with the target gene, and the corresponding protein can be expressed or displayed without continuing to express the GFP gene. The genome of the successfully replaced cells was extracted and primers (RMCE-P1 / P2) were designed to ampl...
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