A kind of cell line for protein display and expression, preparation method and application thereof

Abstract

The present invention provides a CHO cell that can be used for protein display and expression. An expression cassette is integrated into the genome of the CHO cell, and the expression cassette is integrated after the stop codon of the YWHAE gene in the CHO cell genome; the expression cassette Including promoter, first recombinase recognition sequence, target gene, second recombinase recognition sequence, terminator. In this cell, the fluorescent protein gene and antibody gene can be highly transcribed and continuously and stably expressed in the absence of antibiotics, and the antibody transcription level has been significantly improved compared to the previously established CHO-puro cell line. It was verified by experiments that the AID mutation efficiency of this site was significantly higher than that of the random insertion site of CHO-puro.

Description

technical field

[0001] The invention belongs to the field of biomedicine and antibody engineering, and in particular, relates to a cell line for protein display and a preparation method and application thereof. Background technique

[0002] At present, antibodies have been widely used in scientific research, diagnosis and treatment. After the antibody is discovered, the affinity of the antibody is often not high. At this time, in vitro antibody display technology is required to improve the affinity of the antibody so that the antibody can be used. In recent years, mammalian cell antibody display and in vitro hypermutation evolution platforms have developed rapidly.

[0003] In the previous work, the applicant has established a mammalian cell antibody evolution platform, randomly integrated a replaceable cassette expression box into the CHO genome, and obtained a single-copy cell line through multiple rounds of replacement screening and identification CHO-puro (Chinese Pate...

Examples

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...

technical field

[0001] The invention belongs to the field of biomedicine and antibody engineering, and in particular, relates to a cell line for protein display and a preparation method and application thereof. Background technique

[0002] At present, antibodies have been widely used in scientific research, diagnosis and treatment. After the antibody is discovered, the affinity of the antibody is often not high. At this time, in vitro antibody display technology is required to improve the affinity of the antibody so that the antibody can be used. In recent years, mammalian cell antibody display and in vitro hypermutation evolution platforms have developed rapidly.

[0003] In the previous work, the applicant has established a mammalian cell antibody evolution platform, randomly integrated a replaceable cassette expression box into the CHO genome, and obtained a single-copy cell line through multiple rounds of replacement screening and identification CHO-puro (Chinese Pate...

Examples

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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