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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production
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Conversion of MDCK cell line to suspension culture by transfecting with human siat7e gene and its application for influenza virus production

机译:人siat7e基因转染将MDCK细胞系转化为悬浮培养及其在生产流感病毒中的应用

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摘要

MDCK cells are currently being considered as an alternative to embryonated eggs for influenza virus propagation and hemagglu-tinin (HA) production intended for vaccine manufacturing. MDCK cells were found suitable for the virus production but their inability to grow in suspension burdens the process of scale up and hence their production capability. Anchorage-dependent MDCK cells were converted to anchorage-independent cells, capable of growing in suspension as a result of transfection with the human siat7e gene (ST6GalNac V). This gene was previously identified as having an important role in cellular adhesion when the transcriptions of genes from anchorage-dependent and anchorage-independent HeLa cells were compared. Unlike the parental MDCK cells, the sia(7e-expressing cells were capable of growing in shake flasks as suspension cultures, achieving maximum concentration of 7 × 10~5 cells/mL while keeping close to 100% viability throughout the growth phase. In production experiments, the siat7e-expressing cells were infected with the Influenza B/Victoria/504/2000 strain. It was determined that the cell-derived viruses retained similar antigenic properties as those obtained from egg-derived viruses and their nucleotide sequences were identical. The specific production of hemagglutinin (expressed in hemagglutination units per 10~6 cells) from the s/af7e-expressing cells was approximately 20 times higher than the specific production from the parental MDCK cells. If this suspension process scales up, the production potential of HA from 10 L of siat7e-expressing cells at a concentration of 10~6 cells/mL would be equivalent to the amount of HA obtained from 10,000 embryonated eggs.
机译:目前,MDCK细胞被认为可以替代胚胎蛋,用于流感病毒的繁殖和用于疫苗生产的血凝素(HA)的生产。发现MDCK细胞适合于病毒生产,但是它们不能在悬浮液中生长增加了规模扩大的过程,因此增加了它们的生产能力。依赖于锚固的MDCK细胞被转化为不依赖锚固的细胞,由于转染人siat7e基因(ST6GalNac V)而能够悬浮生长。当比较锚定依赖性和非锚定性HeLa细胞的基因转录时,以前已将此基因鉴定为在细胞粘附中具有重要作用。不同于亲代MDCK细胞,表达sia(7e)的细胞能够在摇瓶中作为悬浮培养物生长,达到最大浓度7×10〜5细胞/ mL,同时在整个生长阶段保持接近100%的活力。实验中,将表达siat7e的细胞感染了B / Victoria / 504/2000流感病毒株,确定该细胞衍生的病毒保留了与鸡蛋衍生病毒相似的抗原特性,并且其核苷酸序列相同。来自表达s / af7e的细胞的血凝素的特异性产量(以每10〜6个细胞的血凝单位表示)比亲代MDCK细胞的特异性产量大约高20倍。如果这种悬浮过程扩大规模,HA的生产潜力10 L的siat7e表达细胞中浓度为10〜6细胞/ mL的细胞相当于从10,000个有胚卵中获得的HA量。

著录项

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  • 作者单位

    Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218 Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health;

    Center for Biologics Evaluation and Research, Food and Drug Administration, 9000 Rockville Pike, Bethesda, MD 20892;

    Center for Biologics Evaluation and Research, Food and Drug Administration, 9000 Rockville Pike, Bethesda, MD 20892;

    Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218;

    Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    anchorage-independent; hemagglutinin; sialyltransferase; vaccine;

    机译:独立于锚地;血凝素唾液酸转移酶;疫苗;

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