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Method for increasing human C5 monoclonal antibody expression yield through targeted screening of foreign gene double-integrated locus

An integration site, targeting technology, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as low yield and unstable expression of recombinant proteins

Inactive Publication Date: 2018-11-02
成都金洛克锶生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study using CHO-DUK cells to express human erythropoietin (Epo) found that the integration of the Epo coding gene on large chromosomes (including chromosomes 1 and 2) showed high protein production characteristics, while integration on other chromosomes, especially small chromosomes It will cause unstable or low-yield expression of recombinant protein (Lattenmayer et al., Cytotechnology .2006;51:171–182)

Method used

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  • Method for increasing human C5 monoclonal antibody expression yield through targeted screening of foreign gene double-integrated locus
  • Method for increasing human C5 monoclonal antibody expression yield through targeted screening of foreign gene double-integrated locus
  • Method for increasing human C5 monoclonal antibody expression yield through targeted screening of foreign gene double-integrated locus

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Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Construction and Identification of Antibody Expression Vectors

[0023] According to the published sequence of eculin monoclonal antibody, the light and heavy chain sequences of the synthetic antibody were designed and digested (Avr II and Pac I) and inserted into the modified UCOE expression vector respectively, and then expressed in E. coli to screen the recombinant colonies and extract the plasmid Recombinant plasmids were digested and identified, gene sequence analysis and identification were carried out, and the light and heavy chain eukaryotic expression vectors BP6001M2-2 and BP6002-1 of the antibody were successfully obtained.

Embodiment 2

[0024] Embodiment 2 plasmid transfection and cell selection

[0025] The QIAGEN Plasmid Midi Kit was used to extract the BP6001M2-2 and BP6002-1 transfection plasmids, and the transfection host cell was DG44 (CHO cell line), according to Freedom TM CHO-S TM Kit Kit Instructions Co-transfect the BP6001M2-2 and BP6002-1 plasmids. The cells transferred with the plasmid were placed in a 6-well plate (37° C., 5% CO 2 ) for 48 hours, and the cell viability and cell number were detected by a cell counter.

[0026] After 48 hours of recombinant plasmid transfection, 10nM MTX was used for pressure selection, and the surviving cells were continuously cultured and the concentration of MTX was gradually increased to 500nM; Cultured in an incubator for 2 weeks. When the cells gradually grow to colonies visible to the naked eye, ClonePix FL picks a single colony to prepare monoclonal cells. A total of 90 cell clones with fluorescence MAX>1000 were picked and cloned into 96-well plates...

Embodiment 3

[0027] Embodiment 3 Chromosome and probe preparation

[0028] Add colchicine (final concentration: 1 μg / mL) to the cultured monoclonal cells, continue to incubate at 37°C for 3 hours, collect the cells, and prepare chromosomes according to conventional methods; design specific primers according to the information of the light and heavy chain plasmids, and use The target gene was amplified by PCR and used for probe labeling. Probe labeling and purification were performed according to the instructions of "Random Primed DNA Labeling Kit" (1104760001, Roche) "PCR Purification Kit".

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Abstract

The invention provides a method for increasing human C5 monoclonal antibody expression yield through targeted screening of a foreign gene double-integrated locus. The method comprises the following steps: (1) inserting a light chain encoding gene and a heavy chain encoding gene of a human complement C5 antibody into two eukaryotic expression vectors respectively; (2) acquiring a recombined light chain encoding plasmid and a recombined heavy chain encoding plasmid, and co-transfecting the light chain encoding plasmid and heavy chain encoding plasmid to Chinese hamster ovary (CHO)DG44 cells; (3)screening a light chain encoding gene and a heavy chain encoding gene with a FISH technology, and locating on the cells of a chromosome 1q1 and a chromosome 1q13 by double integration for cloning on.By adopting the method, 1q1 and 1q13 regions obtained by screening support strong transcription of the light chain encoding gene and the heavy chain encoding gene, efficient and stable expression ofthe human C5 monoclonal antibody in the CH0-DG44 cells can be realized, and the product can be used for treating various diseases caused by rise of the C5 level.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for targeting and screening double integration sites to improve the expression yield of human complement C5 monoclonal antibody. Background technique [0002] Human C5 monoclonal antibody ("Soliris", also known as eculizumab) has been approved for "paroxysmal nocturnal hemoglobinuria (PNH)" and "atypical hemolytic uremic syndrome (aHUs)". Other indications such as "refractory myasthenia gravis" have completed clinical trials, and "recurrent neuromyelitis optica spectrum disease" and "antibody-mediated rejection" have also entered the late stage of clinical trials. Eculizumab is currently the most expensive drug in the world. In addition to the special pricing due to the treatment of diseases, early biotechnology and recombinant protein expression systems have restricted the expression and yield of antibodies, which is also an important factor leading to the high ...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/85
CPCC07K16/18C07K2317/76C07K2317/92C07K2317/94C12N15/85
Inventor 高小平张晟代燕平何刚程琳王雯茜付伟
Owner 成都金洛克锶生物技术有限公司
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