| Summary: | 碩士 === 國立中興大學 === 獸醫學系 === 91 === Pseudorabies virus (PrV), a double-stranded DNA virus, belongs to the subfamily Alphaherpesvirinae of neurotropic herpesviruses and is the causative agent of Aujeszky’s disease in swine. PrV encodes information for at least 11 glycoproteins, 10 of which are found in the virion envelope as well as on the surfaces of infected mammalian cells. Glycoprotein H (gH), gL, gB and gD of PrV are not only essential for virus entry at a post-binding stage, but are also sufficient for cell-to-cell spread via fusion with the cellular membranes. Virions that lack glycoprotein H, L, B or D fail to infect cells, thus the PrV glycoprotein H plays an essential role in the fusion of virus envelopes with cellular membranes and in the fusion of an infected cell membranes with the uninfected neighbours. For a detailed analysis of the function of gH, this gene was cloned into prokaryotic and eukaryotic expression systems in this study. The recombinant expression plasmid pETgH was constructed by cloning the gH gene located in the BamH I fragments 11 and 17 of PrV genomic DNA into pET32a(+) vector. The gH protein expressed in E. coli was 76 kDa in size, and the purified product was used as an antigen to immunize mice for generating specific antibodies against viral gH protein. The specificity of immunoserum was identified by recognizing the gH protein in the PrV infected cells in immunoperoxidase staining. Using a transient transfection assay, we have chosen the MMTV-LTR promoter to induce the expression of viral gH gene in mouse-L (LM) cells from several viral promoters, including the CMV and SV40 promoters using chloramphenicol acetyltransferase (CAT) reporter gene assay. In addition, the recombinant eukaryotic expression plasmid pMAMgH that contains the intact gH gene under the control of MMTV-LTR promoter and a neomycin-resistant gene was transfected into LM cells to establish the stable gH-expressing LMgH7 cells. The LMgH7 cells were demonstrated to contain gH gene and stably express gH protein by several analytical methods including PCR, Southern blotting, RT-PCR, Northern blotting and immunoprecipitation. The LMgH7 cells were found to grow at a similar rate as the parental and pMAMneo vector-transfected LM cells. Furthermore, a gH-deletion PrV (gH－PrV) was shown to be capable of penetrating and infecting adjacent LMgH7 cells in contrast to LM cells. These results clearly indicate that gH expressing in cells can provide the gH－PrV as viral glycoprotein H. Thus, the gH protein produced from LMgH7 cells are functional that could displace viral gH protein during infection.
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