Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine

Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has been shown to act as an anticancer tool. Its special feature in inducing apoptosis in cancer cells but not in normal cells enables it to be widely studied for gene and protein therapies. When produ...

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Main Author:	Lim, Shen Ni
Format:	Thesis
Language:	English
Published:	2012
Subjects:	Cancer - Gene therapy Cancer vaccines Tumor proteins
Online Access:	http://psasir.upm.edu.my/id/eprint/32021/1/IB%202012%209R.pdf
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spelling	my-upm-ir.320212015-01-20T08:21:32Z Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine 2012-04 Lim, Shen Ni Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has been shown to act as an anticancer tool. Its special feature in inducing apoptosis in cancer cells but not in normal cells enables it to be widely studied for gene and protein therapies. When produced by in vitro protein synthesis system as a recombinant fusion with maltose-binding protein, the recombinant protein was able to migrate into the nucleus of tumour cells and caused apoptosis in the cells. In principle, minimizing the antigenic regions of a protein could reduce unnecessary side effects of the protein to a cell. The aim of this study was determining the minimal selective domain of Apoptin for apoptotic effect. The full length of Apoptin had been mutated by segmental deletion at the N’ terminal and linking it with nuclear localization sites (NLS1 and NLS2) to develop five truncated constructs. An in vitro protein expression system, RTS was used to express Apoptin and truncated Apoptin proteins. Microinjection method was applied to deliver all the purified proteins into human breast cancer cells, MCF7 and normal human liver cells,Chang cells. The cells were demonstrated for efficient protein delivery when using 2 mg/ml of purified proteins at 60 hPa of injection pressure for 0.5 s. Cellular microinjections of truncated Apoptin proteins, PrVP3A1-69N1N2, PrVP3A1-46N1N2 and PrVP3A1-31N1N2, retained the characteristics of ectopically expressed wild-type full length Apoptin in cancerous cells. Annexin V was used to detect the presence of early apoptosis in the cells and the result showed occurrence of apoptosis in the cell nucleus. All the three truncated complexes were successfully translocated to the nucleus of human breast cancer cells (MCF cells) and induced apoptosis,whereas in normal Chang cells they remained in the cytoplasm with no apparent toxicity. However, two other mutagens, PrVP3A32-69N1N2 and PrVP3A32-62N1N2, induced apoptosis in both cancerous and noncancerous cells, thus lost their specific selectivity to cancerous cells. Compared to the other three truncated Apoptin proteins, both latter proteins had deletion spanning a.a. 1-31, the upper region of leucine-rich sequence (LRS). LRS at the a.a. 33-46 can act as nuclear retention signals. Therefore, the findings suggested that the minimal selective domain for Apoptin resides in the N-terminal specifically within the deletions spanning a.a. 1-33, which could be the leucine-rich sequence (LRS) enhancer and the contributing factor to the non-selectivity of truncated Apoptin proteins, PrVP3A32-69N1N2 and PrVP3A32-62N1N2. This new finding associated to the selectivity of LRS upstream probably could play a role in cancer cells selectivity for apoptosis induction by Apoptin, In conclusion, out of the five truncated Apoptin proteins, three of them (PrVP3A1-69N1N2, PrVP3A1-46N1N2 and PrVP3A1-31N1N2) were functioning similar to the full length wild type Apoptin, in their ability to induce apoptosis selectively to the targeted cancer cells. Cancer - Gene therapy Cancer vaccines Tumor proteins 2012-04 Thesis http://psasir.upm.edu.my/id/eprint/32021/ http://psasir.upm.edu.my/id/eprint/32021/1/IB%202012%209R.pdf application/pdf en public masters Universiti Putra Malaysia Cancer - Gene therapy Cancer vaccines Tumor proteins Institute of Bioscience
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Cancer - Gene therapy Cancer vaccines Tumor proteins
spellingShingle	Cancer - Gene therapy Cancer vaccines Tumor proteins Lim, Shen Ni Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
description	Chicken Anemia Virus (CAV) VP3 protein (also known as Apoptin), a basic and proline-rich protein has been shown to act as an anticancer tool. Its special feature in inducing apoptosis in cancer cells but not in normal cells enables it to be widely studied for gene and protein therapies. When produced by in vitro protein synthesis system as a recombinant fusion with maltose-binding protein, the recombinant protein was able to migrate into the nucleus of tumour cells and caused apoptosis in the cells. In principle, minimizing the antigenic regions of a protein could reduce unnecessary side effects of the protein to a cell. The aim of this study was determining the minimal selective domain of Apoptin for apoptotic effect. The full length of Apoptin had been mutated by segmental deletion at the N’ terminal and linking it with nuclear localization sites (NLS1 and NLS2) to develop five truncated constructs. An in vitro protein expression system, RTS was used to express Apoptin and truncated Apoptin proteins. Microinjection method was applied to deliver all the purified proteins into human breast cancer cells, MCF7 and normal human liver cells,Chang cells. The cells were demonstrated for efficient protein delivery when using 2 mg/ml of purified proteins at 60 hPa of injection pressure for 0.5 s. Cellular microinjections of truncated Apoptin proteins, PrVP3A1-69N1N2, PrVP3A1-46N1N2 and PrVP3A1-31N1N2, retained the characteristics of ectopically expressed wild-type full length Apoptin in cancerous cells. Annexin V was used to detect the presence of early apoptosis in the cells and the result showed occurrence of apoptosis in the cell nucleus. All the three truncated complexes were successfully translocated to the nucleus of human breast cancer cells (MCF cells) and induced apoptosis,whereas in normal Chang cells they remained in the cytoplasm with no apparent toxicity. However, two other mutagens, PrVP3A32-69N1N2 and PrVP3A32-62N1N2, induced apoptosis in both cancerous and noncancerous cells, thus lost their specific selectivity to cancerous cells. Compared to the other three truncated Apoptin proteins, both latter proteins had deletion spanning a.a. 1-31, the upper region of leucine-rich sequence (LRS). LRS at the a.a. 33-46 can act as nuclear retention signals. Therefore, the findings suggested that the minimal selective domain for Apoptin resides in the N-terminal specifically within the deletions spanning a.a. 1-33, which could be the leucine-rich sequence (LRS) enhancer and the contributing factor to the non-selectivity of truncated Apoptin proteins, PrVP3A32-69N1N2 and PrVP3A32-62N1N2. This new finding associated to the selectivity of LRS upstream probably could play a role in cancer cells selectivity for apoptosis induction by Apoptin, In conclusion, out of the five truncated Apoptin proteins, three of them (PrVP3A1-69N1N2, PrVP3A1-46N1N2 and PrVP3A1-31N1N2) were functioning similar to the full length wild type Apoptin, in their ability to induce apoptosis selectively to the targeted cancer cells.
format	Thesis
qualification_level	Master's degree
author	Lim, Shen Ni
author_facet	Lim, Shen Ni
author_sort	Lim, Shen Ni
title	Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
title_short	Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
title_full	Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
title_fullStr	Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
title_full_unstemmed	Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine
title_sort	truncated chicken anemia virus-vp3 as a potential anti tumour vaccine
granting_institution	Universiti Putra Malaysia
granting_department	Institute of Bioscience
publishDate	2012
url	http://psasir.upm.edu.my/id/eprint/32021/1/IB%202012%209R.pdf
_version_	1747811642461126656

Truncated chicken anemia virus-VP3 as a potential anti tumour vaccine

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