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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| Format: | Thesis |
| Language: | English |
| Published: |
2012
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/32021/1/IB%202012%209R.pdf |
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| Summary: | 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. |
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