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目的:研究单纯疱疹病毒Ⅱ型(HSV-2)潜伏相关转录体(LAT)开放读码框1(ORF1)的表达特点及其对Vero细胞活性的影响。方法:双酶切和测序验证本实验室构建的HSV-2 LAT ORF1真核表达载体pEGFP-ORF1,并以转染试剂盒Xfect介导其转染至Vero细胞,通过RT-PCR和绿色荧光蛋白检验其在细胞中的表达,用MTT法进行细胞活性分析。结果:重组质粒表达的融合蛋白主要集中细胞核,而空质粒表达的绿色荧光蛋白在细胞核和细胞质中分布均匀;重组质粒对Vero细胞没有损伤作用。结论:HSV-2 LAT ORF1影响了绿色荧光蛋白的分布,可降低空质粒对细胞的损伤作用;其作用位点可能主要定位在细胞核中,为阐明HSV-2 LAT ORF1在潜伏复发中的功能奠定了实验基础。
Objective: To investigate the expression of open reading frame 1 (ORF1) of herpes simplex virus type 2 (HSV-2) latent-related transcript (LAT) and its effect on Vero cell activity. Methods: The eukaryotic expression vector pEGFP-ORF1 of HSV-2 LAT ORF1 constructed by our laboratory was verified by double enzyme digestion and sequencing. The vector was transfected into Vero cells by Xfect and was transfected into Vero cells by RT-PCR and GFP The expression in cells was examined, and cell viability was analyzed by MTT method. Results: The fusion protein expressed in the recombinant plasmid mainly concentrated in the nucleus, whereas the green fluorescent protein expressed in the empty plasmid was evenly distributed in the nucleus and cytoplasm. The recombinant plasmid had no effect on Vero cells. CONCLUSIONS: HSV-2 LAT ORF1 affects the distribution of green fluorescent protein (GFP) and decreases the damage effect of empty plasmid on cells. The site of HSV-2 LAT ORF1 may mainly locate in the nucleus. To clarify the function of HSV-2 LAT ORF1 in latent relapse The experimental basis.