Development of a recombinant CHO cell line
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Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2017
Författare
Berglund, Martina
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Anti-Müllerian hormone (AMH) is biological marker for the ovarian reserve, independent of the menstrual cycle. The AMH serum level can be measured with immunoassay in order to predict premature ovarian failure, chance for success of in vitro fertilization and aid in the diagnosis of polycystic ovary syndrome (PCOS). The aim of the thesis was to develop a CHO cell line with overexpression of recombinant human AMH. Characteristics of the produced AMH were investigated in order to evaluate suitability for use in calibrators for an immunoassay. CHO cells are well established safe hosts for production of recombinant protein and have numerous of advantages such as capacity for efficient post-translational modifications and easy adaptation to growth in serum free medium. The experiments were held out with two different CHO cell lines and two different vectors, one with codon optimized AMH and one with the native human AMH sequence. Two different transfection methods were used, chemically based and electroporation followed by two different selection systems. The results of the thesis show that a stable recombinant CHO cell line could be established for AMH by transfection of a vector containing the native AMH sequence. The results also indicated that the C-terminal purification tag in the expression vector altered expression. Selection with G418 enabled establishment of clones with stable expression. The produced AMH had desirable stability and properties like native AMH present in serum and plasma. This indicates that the produced AMH could be suitable for use in calibrators for immunoassay measuring native human AMH.
Beskrivning
Ämne/nyckelord
Livsvetenskaper , Annan industriell bioteknik , Cell- och molekylärbiologi , Medicinsk bioteknologi (med inriktning mot cellbiologi) , Life Science , Other Industrial Biotechnology , Cell and Molecular Biology , Medical Biotechnology (with a focus on Cell Biology)