Characterization of Fusion Protein Composed of Herpes Simplex Virus VP22 Protein and Cytosine Deaminase
| Autor: | Chu Mei-Hung, 朱美虹 |
|---|---|
| Rok vydání: | 2001 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 89 Gene-directed enzyme prodrug therapy (GDEPT) is based on the delivery of a gene that encodes an enzyme, which is non-toxic per se, but is able to convert a prodrug into a potent cytotoxin. The bacteria cytosine deaminase (BCD) can convert the non-toxic 5’-fluorocytosine (5-FC) to 5’-fluorouracil (5-FU). A recently discovered yeast cytosine deaminase (YCD) possesses similar enzymatic activity, but it is 30-fold higher than the BCD. The Herpes Simplex Virus (HSV) VP22 protein is a special protein, which can translocate among cells. Once synthesized in infected cells, VP22 spreads efficiently via a Golgi-independent pathway to surrounding uninfected cells, where it specifically accumulates in the nucleus. This study investigates the possibility of fusing the cytosine deaminase with the VP22 in order to enhance the bystander effect. Single genes, EGFP, BCD or YCD and fusion genes composed of VP22 and EGFP, BCD or YCD were constructed. The constructs were transfected into 293T cells. Expression of fusion protein or individual protein was examined by fluorescence microscopy or immunoflourescence staining. It was found that the VP22EGFP, VP22BCD and VP22YCD were present in the cytoplasm and the nucleus of the transfected cells, 293T. However, EGFP, BCD and YCD transfected cell were present only in the cytoplasm of the transfected cells. This phenomenon suggested the VP22 might have the ability to traffick from cell to cell. To further verify that VP22 can enhance the bystander effect of BCD and YCD, we co-cultured effector cells transfected BCD, YCD, VP22BCD, VP22YCD and VP22 with blank cells at different ratios. The mixture was then treated with 5-FC and assayed by WST-1. The result demonstrate that, when co-cultured with CT-26 cells, the killing effect of VP22YCD was better then that of YCD. Yet, there is no significant difference between those of VP22BCD and BCD. In contrast, when effector cells were co-cultured with GP7TB cells, VP22BCD possessed better killing effect than BCD. No significant difference between VP22YCD and YCD was observed, probably due to the high killing activity. The different outcomes may be resulted from different sensitivity of GP7TB and CT-26 cells to the 5-FU, and also from the different enzyme activities of YCD and BCD. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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