20.500.12556/DiRROS-20012 TRIM28 and [beta]-actin identified via nanobody-based reverse proteomics approach as possible human glioblastoma biomarkers Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls. malignant gliomas cancer stem cells nanobodies maligni gliomi izvorne rakave celice nano telesa true false true Angleški jezik Slovenski jezik Neznano 2024-08-02 08:48:06 2024-08-02 08:48:07 2024-08-03 03:45:58 0000-00-00 00:00:00 2014 0 0 Nasl. z nasl. zaslona; Opis vira z dne 25. 11. 2014; Članek v pdf obsega 22 str.; iss. 11 Vol. 9 Nov. 2014 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2014-11-24 620.3 1932-6203 10.1371/journal.pone.0113688 3251791 http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0113688 1 6b30321b-508a-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27833 RAZ_Jovchevska_Ivana_2014.pdf RAZ_Jovchevska_Ivana_2014.pdf 1 AA68A2E044D5298AA9A30E5A88CB9306 c0dda4de47a0430a914497d8b94d5617ff929d01d2b46f96be35ec7dca679568 351766f7-508b-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27834 https://doi.org/10.1371/journal.pone.0113688 0 6d040613-508b-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=27836 Nacionalni inštitut za biologijo 0 0 0