1. Expression of fluorescent tagged recombinant erythroferrone protein
Min Min THAN ; Jetsada RUANGSURIYA ; Somdet SRICHAIRATANAKOOL ; Chairat UTHAIPIBULL ; Min Min THAN
Asian Pacific Journal of Tropical Biomedicine 2018;8(7):360-364
Objective: To produce fluorescent tagged recombinant erythroferrone protein (ERFE-eGFP) for laboratory investigations. Methods: Erythroferrone (ERFE) gene was fused to green fluorescent protein (eGFP) gene and cloned in a pSecTag2Hygro plasmid. The constructed plasmid was amplified in Escherichia coli DH5 α and the eGFP-fused ERFE (ERFE-eGFP) protein was expressed in human embryonic kidney (HEK293T) cell line. Results: The plasmid constructed from colony C6 contained ERFE-eGFP with the correct restriction sizes of 4.2 kb and expressed secretory ERFE-eGFP fusion protein (approximately size of 75 kDa) in HEK293T cell line. Conclusions: ERFE-eGFP recombinant protein is successfully expressed as a secretory functional protein and could be sensitively detected using fluorometry. This fusion protein might benefit future applications for localization of cellular ERFE receptors and competitive immunoassay of ERFE concentration.
2.Alpha-Type 1 Polarized Dendritic Cells Loaded with Apoptotic Allogeneic Breast Cancer Cells Can Induce Potent Cytotoxic T Lymphocytes against Breast Cancer.
Min Ho PARK ; Deok Hwan YANG ; Mi Hyun KIM ; Jae Hong JANG ; Yoon Young JANG ; Youn Kyung LEE ; Chun Ji JIN ; Than Nhan Nguyen PHAM ; Truc Anh Nguyen THI ; Mi Seon LIM ; Hyun Ju LEE ; Cheol Yi HONG ; Jung Han YOON ; Je Jung LEE
Cancer Research and Treatment 2011;43(1):56-66
PURPOSE: Various tumor antigens can be loaded onto dendritic cells (DCs) to induce a potent cytotoxic T lymphocyte (CTL) response in DC-based immunotherapy against breast cancer. However, in the clinical setting, obtaining a sufficient number of autologous tumor cells as a source of tumor antigens is a laborious process. We therefore investigated the feasibility of immunotherapy using breast-cancer-specific CTLs generated in vitro by use of alpha-type 1 polarized DCs (alpha DC1s) loaded with ultraviolet B-irradiated cells of the breast cancer cell line MCF-7. MATERIALS AND METHODS: alphaDC1s were induced by loading allogeneic tumor antigen generated from the MCF-7 UVB-irradiated breast cancer cell line. Antigen-pulsed alphaDC1s were evaluated by morphological and functional assays, and the breast-cancer-specific CTL response was analyzed by cytotoxic assay. RESULTS: The alphaDC1s significantly increased the expression of several molecules related to DC maturation without differences according to whether the alphaDC1s were loaded with tumor antigens. The alphaDC1s showed a high production of interleukin-12 both during maturation and after subsequent stimulation with CD40L, which was not significantly affected by loading with tumor antigens. Breast-cancer-specific CTLs against autologous breast cancer cells were successfully induced by alphaDC1s loaded with apoptotic MCF-7 cells. CONCLUSION: Autologous DCs loaded with an allogeneic breast cancer cell line can generate potent breast-cancer-specific CTL responses. This may be a practical method for cellular immunotherapy in patients with breast cancer.
Antigens, Neoplasm
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Breast
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Breast Neoplasms
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CD40 Ligand
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Cell Line
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Dendritic Cells
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Humans
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Immunotherapy
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Interleukin-12
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Lymphocytes
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T-Lymphocytes, Cytotoxic