Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells

Recombinant antibody cloning and phage display technologies can be employed to produce and isolate single-chain antibodies (scFv) specifically against antigen of interest. The aims of this study are to construct single chain variable fragment (scFv) towards MCF-7 breast cancer cells and to character...

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Main Author:	Ahmad, Zuhaida Asra
Format:	Thesis
Language:	English English
Published:	2008
Subjects:	Breast - Cancer Diseases
Online Access:	http://psasir.upm.edu.my/id/eprint/5388/1/IB_2008_1.pdf
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id	my-upm-ir.5388
record_format	uketd_dc
spelling	my-upm-ir.53882013-05-27T07:22:28Z Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells 2008 Ahmad, Zuhaida Asra Recombinant antibody cloning and phage display technologies can be employed to produce and isolate single-chain antibodies (scFv) specifically against antigen of interest. The aims of this study are to construct single chain variable fragment (scFv) towards MCF-7 breast cancer cells and to characterize scFv antibodies that interacts with MCF-7. Initially, mRNA was extracted from previously well-characterized monoclonal antibody (C3A8) against MCF-7. The genes encoding heavy (VH) and light (VL) chains were amplified, linked in VH-VL orientation via PCR and cloned into a pCANTAB 5E phagemid vector. The protein was then expressed in a supE strain of E. coli TG1. Phage particles displaying scFv were panned against MCF-7 and the selected clones were further used for infecting non-suppressor strain, E. coli HB2151. The scFv antibodies expressed were characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. As demonstrated by ELISA result, the scFv antibodies could strongly bind to MCF-7 breast cancer cells. It retained the binding capacity of its parental C3A8 monoclonal antibody. Clone B7 was expressed mainly as soluble periplasmic protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 32 kDa. Nucleotide sequence analysis of C3A8 scFv showed high homology (99%) with published single chain antibody against rice stripe virus protein P20 [synthetic construct]. In conclusion, the recombinant antibody technology is an effective approach in the development of scFv antibody for the next generation of immunotherapy reagents especially towards MCF-7 breast cancer cells. Breast - Cancer Diseases 2008 Thesis http://psasir.upm.edu.my/id/eprint/5388/ http://psasir.upm.edu.my/id/eprint/5388/1/IB_2008_1.pdf application/pdf en public masters Universiti Putra Malaysia Breast - Cancer Diseases Institute Bioscience English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Breast - Cancer Diseases
spellingShingle	Breast - Cancer Diseases Ahmad, Zuhaida Asra Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
description	Recombinant antibody cloning and phage display technologies can be employed to produce and isolate single-chain antibodies (scFv) specifically against antigen of interest. The aims of this study are to construct single chain variable fragment (scFv) towards MCF-7 breast cancer cells and to characterize scFv antibodies that interacts with MCF-7. Initially, mRNA was extracted from previously well-characterized monoclonal antibody (C3A8) against MCF-7. The genes encoding heavy (VH) and light (VL) chains were amplified, linked in VH-VL orientation via PCR and cloned into a pCANTAB 5E phagemid vector. The protein was then expressed in a supE strain of E. coli TG1. Phage particles displaying scFv were panned against MCF-7 and the selected clones were further used for infecting non-suppressor strain, E. coli HB2151. The scFv antibodies expressed were characterized by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. As demonstrated by ELISA result, the scFv antibodies could strongly bind to MCF-7 breast cancer cells. It retained the binding capacity of its parental C3A8 monoclonal antibody. Clone B7 was expressed mainly as soluble periplasmic protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 32 kDa. Nucleotide sequence analysis of C3A8 scFv showed high homology (99%) with published single chain antibody against rice stripe virus protein P20 [synthetic construct]. In conclusion, the recombinant antibody technology is an effective approach in the development of scFv antibody for the next generation of immunotherapy reagents especially towards MCF-7 breast cancer cells.
format	Thesis
qualification_level	Master's degree
author	Ahmad, Zuhaida Asra
author_facet	Ahmad, Zuhaida Asra
author_sort	Ahmad, Zuhaida Asra
title	Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
title_short	Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
title_full	Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
title_fullStr	Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
title_full_unstemmed	Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells
title_sort	construction of a single chain variable fragment against mcf-7 breast cancer cells
granting_institution	Universiti Putra Malaysia
granting_department	Institute Bioscience
publishDate	2008
url	http://psasir.upm.edu.my/id/eprint/5388/1/IB_2008_1.pdf
_version_	1747810413579337728

Construction of a Single Chain Variable Fragment Against Mcf-7 Breast Cancer Cells

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