| Summary: | 碩士 === 國立成功大學 === 微生物暨免疫學研究所 === 87 === The envelope glycoprotein gp50 of pseudorabies virus (PRV) is essential for virus entry, but is not required for subsequent steps in the viral replication cycle. Phenotypically-complemented gp50 null mutants can infect cells and can spread, both in vitro and in vivo, by direct cell-to-cell transmission. However, progeny virions released by the infected cells are non-infectious because they lack gp50. The aim of this study is to construct gp50-defective PRV vectors for the applications of recombinant vaccines and gene therapy. The transfer vectors carrying the genes encoding either herpes simplex virus thymidine kinase (HSV-TK) or enhanced green fluorescent protein (EGFP) were designed to replace the gp50 gene. By homologous recombination, NIH3T3 cells expressing PRV gp50 were cotransfected with the transfer vector and the TK-/gI- PRV that is defective in both viral TK and gI, a nonessential glycoprotein. The recombinant PRV carrying HSV-TK, but defective in gp50 and gI, was isolated by selection with hypoxanthine, aminopterin and thymidine (HAT) media, and the HSV-TK gene was also detectable by PCR in the viral supernatant. The plaque size produced by gp50-defective PRV was bigger than that produced by parental TK-/gI- PRV. Moreover, the former was more sensitive than the latter to acyclovir. Mice treated intraperitoneally with gp50-/HSV-TK+ or parental TK-/gI- PRV have prolonged survival time compared with those treated with wild-type PRV. These results suggest that gp50-defective PRV may be exploited for the development of recombinant PRV vaccines. A similar strategy was used to replace the HSV-TK gene with the EGFP gene following acyclovir selection. The EGFP gene was detected by PCR in the viral supernatant. The expression of EGFP in NIH3T3/gp50 cells infected with TK-/gI-/gp50-/EGFP+ virus was examined using a standard fluorescein isothiocyanate (FITC) filter-equipped fluorescence microscope. Taken together, the gp50-defective PRV may be explored as potential mammalian expression vectors for recombinant vaccines and for gene therapy.
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