禽类多瘤病毒APV-1晚期基因多顺反子的EGFP标记载体构建和表达  

Construction and Expression of the Polycistronic Vector of Avian Polyomavirus 1 Late Genes Tagged with EGFP

作　　者：李劲[1] 刘琳[1] 

机构地区：[1]中南民族大学生命科学学院,武汉430074

出　　处：《中南民族大学学报（自然科学版）》2016年第1期39-43,共5页Journal of South-Central University for Nationalities：Natural Science Edition

基　　金：教育部留学回国人员科研启动基金[教外司留(2007)1108号]

摘　　要：为研究禽类多瘤病毒晚期基因多顺反子翻译起始调控的机制,设计和构建了以增强绿色荧光蛋白(EGFP)报告基因替代病毒APV-1野生型的晚期结构蛋白VPs基因的真核双顺反子表达载体,并观察其在转染进入禽类原代成纤维细胞中的表达翻译状态.以APV-1的c DNA克隆(p HL1003)为模板,运用PCR技术扩增出与野生型病毒APV-1相同的Ori区、早期编码区和晚期Agno-1a区序列作为载体片段;从质粒p EGFP-N1中扩增出编码绿色荧光的EGFP DNA片段;经连接构成重组质粒p HL1003-GFP,通过脂质体细胞转染方法将质粒DNA转染到鸡胚胎和鹌鹑胚胎成纤维细胞中,通过细胞培养观察荧光表达状况.DNA序列分析证实了重组克隆中的EGFP序列与已知的质粒p EGFP-N1中EGFP序列一致.转染72 h后观察鸡和鹌鹑胚胎成纤维细胞,转染成功胚胎细胞明显表达较强的荧光,说明GFP可以在构建的双顺反子重组质粒的下游中正常表达并产生荧光.p HL1003-GFP重组质粒的构建及其在禽类原代成纤维细胞中的高效表达,为我们研究多顺反子翻译起始调控中Agno-1a基因的表达对下游病毒结构蛋白基因的翻译调控机制提供了简洁易用的系统和模型.To study the mechanism of translational initiation regulation of the polycistronic mRNA of the Avain Polyomavirus late genes, the eukaryotic expression vector with bi-cistronic mRNA was designed and constructed, in which the downstream late structural proteins of wild-type APV-1 were replaced by EGFP reporter gene. By transfection of the recombinant DNA into the bird primary fibroblast cells, the expression of EGFP was observed. Using APV-1 cDNA plasmid as template, the early and Ori regions including Agno-1a gene of the viral genome were amplified by PCR. The EGFP coding sequence was amplified from plasmid pEGFP-N1. After ligation of the above two kinds of DNA fragments, the recombinant plasmid pHL1003-GFP was identified by restriction enzyme digestions, PCR and DNA sequencing. The viral recombinant DNA was transfected into chicken embryo and quail embryo fibroblasts by lipofection respectively. The EGFP DNA sequence of the recombinant pHL1003-GFP was consistent with the EGFP sequence in the pEGFP-N1 plasmid by DNA sequencing analysis. After 72 h transfection , the GFP gene at downstream of the bi-cistronic mRNA was able to be expressed effectively, and the strong fluorescence of the GFP was excited in chicken embryo and quail embryo fibroblast cells. The construction of pHL1003-GFP recombinant plasmid and its highly expressed GFP with strong capability of fluorescence excitation in avian primary fibroblast cells provide a simple and easy testing system and an experimental model to explore the translational regulation mechanism in poly-cistronic mRNA of the Agno-1a gene for its downstream genes expression, which coding APV-1 late structural proteins.

关 键 词：禽类多瘤病毒 增强绿色荧光蛋白 重组质粒 转染 表达 

分 类 号：Q291[生物学—细胞生物学]