Cell strain for protein display and expression as well as preparation method and application thereof

A protein display and cell technology, which is applied in the field of protein display cell lines and its preparation, can solve the problems of affecting RNA polymerase II transcription activity, complex chromosome structure, and difficulties

Active Publication Date: 2020-04-10
HYQUO MOLECULE BEIJING TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chromosome structure is relatively complex, which can affect the transcriptional activity of RNA polymerase II, and the dynamic structure...

Method used

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  • Cell strain for protein display and expression as well as preparation method and application thereof
  • Cell strain for protein display and expression as well as preparation method and application thereof
  • Cell strain for protein display and expression as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment one, material and method

[0030] 1. Cell culture

[0031] Both CHO-K1 cells and a series of cells constructed using CHO-K1 use PRMI-1640 medium containing 10% fetal bovine serum (Hyclone), 100U / mL double antibody, at 37°C, 5% CO 2 cultured in an incubator. CHO / dhFr- (dihydrofolate reductase-deficient Chinese hamster ovary cells) used IMDM medium containing 10% fetal bovine serum (Hyclone), 100U / mL double antibody, 0.1mM hypoxanthine and 0.016mM thymine, in 37°C, 5% CO 2 cultured in an incubator.

[0032] Human 293F cells using FreeStyle TM 293Expression Medium at 37°C, 5% CO 2 Incubator, shake flask culture at 120rpm rotation speed.

[0033] 2. Cell transfection and establishment of cell lines

[0034] In order to construct a stable cell line with specific site integration, CHO-K1 cells were seeded into 6-well plates one day in advance, and when the cells expanded to 60%-80% fullness the next day, they were mixed with 500ng donor plasmid and 500ng CRI...

Embodiment 2

[0045] Example 2. Construction of CHO cell lines for protein display

[0046] In the present invention, a replacement expression frame is inserted into a specific site in the CHO cell genome, thereby constructing a high-expression cell line of a single-copy gene. This cell line can quickly replace any antibody gene or other genes into the genome, realize high transcription and expression of antibody genes and display antibodies on the surface of CHO cell membranes for affinity maturation of antibodies.

[0047] In the present invention, the YWHAE gene of the CHO cell genome is selected as the site-specific insertion position of the alternative expression cassette, and three sites of the YWHAE gene are selected for gene-site insertion of the alternative expression cassette. These three sites are E site (exon1), I site (intron, between exon 3 and exon 4) and T site (behind the stop codon of YWHAE gene), such as figure 1 shown.

[0048] The inserted alternative expression cassett...

Embodiment 3

[0052] Example 3, double-combinase-mediated target gene frame replacement

[0053] In order to detect whether the obtained CHO-K1-T cell line can perform double histone-mediated frame replacement and the corresponding protein expression (in the following embodiment, the CHO-K1-T cell line can also be used T31 or replace). We replaced the GFP gene in the clone with the 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. The sorted cells were cultured and expanded, and collected for flow analysis. The representative results are as follows: figure 2 shown.

[0054] Such as figure 2 As shown, the GFP gene in CHO-K1-T can be successfully replaced with the target gene, and the corresponding protein is 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 t...

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Abstract

The invention provides a CHO cell for protein display and expression, an expression cassette is integrated in a genome of the CHO cell, and the expression cassette is integrated behind a termination codon of a YWHAE gene of the genome of the CHO cell; the expression cassette comprises a promoter, a first recombinase recognition sequence, a target gene, a second recombinase recognition sequence anda terminator. In the cell, the fluorescent protein gene and the antibody gene can be highly transcribed and continuously and stably highly expressed under the condition of no antibiotic maintenance,and the antibody transcription level is remarkably improved compared with that of a CHO-puro cell strain established before. The experiments prove that the AID mutation efficiency of the site is significantly higher than that of a random insertion site of CHO-puro.

Description

technical field [0001] The invention belongs to the fields of biomedicine and antibody engineering, and in particular relates to a cell line for protein display and its preparation method and application. Background technique [0002] At present, antibodies have been widely used in the fields of 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 needed to improve the affinity of the antibody so that the antibody can be applied. In recent years, mammalian cell antibody display and in vitro high-frequency mutation evolution platforms have been developed rapidly. [0003] In previous work, the applicant has established a mammalian cell antibody evolution platform, randomly integrated an alternative cassette expression cassette into the CHO genome, and obtained a single-copy cell line through multiple rounds of replacement screening CHO-puro (Chine...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85
CPCC12N5/0682C12N15/85C12N2800/60C12N2800/30C12N2830/36C12N2510/00
Inventor 赵云范英俊安莉莉杭海英
Owner HYQUO MOLECULE BEIJING TECH CO LTD
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