摘要：

旨在对羊口疮病毒(ORFV)ORFV113蛋白进行转录动力学分析、真核表达及亚细胞定位研究。使用DNAStar软件对ORFV-JS株ORFV113基因进行序列比对分析;在阿糖胞苷(Arac)存在(Arac+)或不存在(Arac-)的情况下,于ORFV感染Hela细胞后多个时间点(2,4,6,12,24 h)收获细胞,提取细胞总RNA,逆转录 PCR(RT-PCR)扩增ORFV113基因,确定ORFV113的动态转录水平;以ORFV-JS株基因组为模板,PCR扩增ORFV113基因,将其亚克隆到真核表达载体pEGFP-N1中,构建pEGFP-ORFV113重组质粒,经酶切、测序鉴定正确后,使用脂质体Lipofectamine 3000瞬时转染pEGFP-N1质粒和pEGFP-ORFV113重组质粒至HEK293细胞,通过Western Blotting 鉴定ORFV113-EGFP融合蛋白的表达;将pEGFP-N1质粒和pEGFP-ORFV113重组质粒瞬时转染Hela细胞,24 h后使用Hoechst 33342对细胞核染色,倒置荧光显微镜下观察ORFV113蛋白的亚细胞定位。结果表明,ORFV-JS株ORFV113基因全长627 bp,在ORFV毒株中高度保守,属于ORFV早期基因;成功构建了pEGFP-ORFV113重组质粒,ORFV113-EGFP融合蛋白在HEK293细胞中成功表达,大小为60~70 ku,比预测分子量大10~20 ku,预示ORFV113蛋白发生了翻译后修饰;亚细胞定位研究表明,ORFV113蛋白主要定位在Hela细胞的胞质中。综上,本研究成功地对ORFV113蛋白进行了转录动力学分析、真核表达及亚细胞定位。

关键词: 羊口疮病毒, ORFV113蛋白, 转录动力学, 真核表达, 亚细胞定位

Abstract:

In order to perform transcription kinetics,eukaryotic expression and subcellular localization of ORFV113 protein.DNAStar software was used to perform alignment analysis of ORFV113 gene.In the presence or absence of cytarabine,Hela cells were harvested and the total RNA were extracted at various time points(2,4,6,12,24 h)post ORFV infection.RT-PCR was used to amplify ORFV113 gene to determine the dynamic transcription level of ORFV113 gene.ORFV113 gene was amplified by PCR and subcloned into pEGFP-N1 vector to construct pEGFP-ORFV113 recombinant plasmid.After being correctly identified by restriction enzyme digestion and Sanger sequencing,the pEGFP-N1 plasmid and pEGFP-ORFV113 plasmid was transiently transfected into HEK293 cells with Lipofectamine 3000.The expression of ORFV113-EGFP fusion protein in HEK293 cells was detected by Western Blot.Hela cells were transiently transfected with pEGFP-N1 plasmid and pEGFP-ORFV113 recombinant plasmid.After 24 h,the nucleus was stained with Hoechst 33342,and the subcellular localization of ORFV113 protein was observed under an inverted fluorescence microscope.The results revealed that ORFV113 gene was 627 bp in length,which was highly conserved among ORFV strains.It belonged to an early gene of ORFV.The pEGFP-ORFV113 recombinant plasmid was successfully constructed.The ORFV113-EGFP fusion protein with a molecular mass between 60 and 70 ku,approximately 10—20 ku larger than the predicted molecular mass,was successfully expressed in HEK293 cells,indicating that ORFV113 protein had undergone post-translational modification.ORFV113 protein mainly localized to the cytoplasm of Hela cells.In conclusion,the transcription kineics,eukaryotic expression and subcellular localization of ORFV113 protein were successfully performed in the present study.

Key words: ORFV, ORFV113 protein, Transcription kinetics, Eukaryotic expression, Subcellular localization