دورية أكاديمية
Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus
| العنوان: | Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus |
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| المؤلفون: | A. L. Matveev, Ya. A. Khlusevich, I. K. Baykov, I. V. Babkin, E. P. Goncharova, V. V. Morozova, N. V. Tikunova |
| المصدر: | Вавиловский журнал генетики и селекции, Vol 21, Iss 8, Pp 993-1000 (2018) |
| بيانات النشر: | Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders, 2018. |
| سنة النشر: | 2018 |
| المجموعة: | LCC:Genetics |
| مصطلحات موضوعية: | fully human antibody, producing strain, cho cells, plasmid, genomic amplification, ectromelia virus, Genetics, QH426-470 |
| الوصف: | Fullyhuman antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of fullsize antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fullyhuman antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHOS. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFRCHCL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRTDHFRCHCL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRTDHFRCHCL cassette plasmid. Then, CHOS/FRT cells, which contain the FRTsite for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRTsite, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated antivaccinia virus activity in the plaque reduction neutralization test in vitro. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English Russian |
| تدمد: | 2500-3259 |
| Relation: | https://vavilov.elpub.ru/jour/article/view/1279; https://doaj.org/toc/2500-3259 |
| DOI: | 10.18699/VJ17.324 |
| URL الوصول: | https://doaj.org/article/a5b7246d7aaa48068517dbcd4e75a477 |
| رقم الأكسشن: | edsdoj.5b7246d7aaa48068517dbcd4e75a477 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 25003259 |
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| DOI: | 10.18699/VJ17.324 |