Development of Monoclonal Antibody Against Canine Heat Shock Protein 27
| Autor: | Cheng-Wei Liao, 廖振瑋 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 Canines have been regarded as a suitable animal model of human cancers. Heat shock protein B1/27(HSP B1/27) is a member of the small chaperone family, whose functions are critical to the proper folding of proteins and the protection of cells from stress-induced damages. In breast cancer, overexpression of HSP B1/27 is associated with increased cancer progression, increased invasiveness, and resistance to chemotherapeutic drugs. This research focuses on the production and application of monoclonal antibody against canine HSP B1/27 (cHSP B1/27) protein. The full-length cDNA of cHSP B1/27 was amplified by RT-PCR from the total RNA of DTK-SME cells, a canine breast cancer cell line. The amplified 618-bp fragment was then inserted into expression vector pET24a, and the ligation product was used to transform E.coli BL21(DE3) cells. The HSP B1/27 recombinant protein was obtained from the transformed BL21 cells by IPTG-induction, and was further purified by Ni-affinity column. To produce cHSP B1/27-specific antibody, the purified recombinant cHSP B1/27 protein was used as immunogene to immunize mice. 8 weeks post-immunization, the production of specific polyclonal antibodies against cHSP B1/27 was confirmed in the immunized mice, from whom spleen cells were collected to fuse with mouse myeloma cells (NS-1) to generate cHSP B1/27-specific monoclonal antibody. To demonstrate the potential application of the cHSP B1/27 monoclonal antibody, the antibody was utilized to detect the sub-cellular localization of cHSP B1/27 by immunofluorescence microscopy. Our data showed that cHSP B1/27 mainly distributed in the cytoplasm of DTK-SME cells. In this thesis, high-efficiency monoclonal antibody against cHSP B1/27 was successfully prepared. We expect this antibody would benefit future researches on the breast cancer in canines and humans. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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