| 摘要: |
| [目的]为了提高马传染性贫血病毒(EIAV)基因转移载体质粒pcPPTPRE(+)的外源蛋白表达能力,对其转录后调控元件进行优化.[方法]将PCR扩增土拨鼠肝炎病毒(Woodchuck Hepatitis Virus,WHV)转录后调控元件(WPRE),克隆于pGM-T载体获得重组质粒pGM-WPRE,利用NotⅠ酶将WPRE亚克隆入pcPPTPRE(+),筛选WPRE正向连接的重组质粒pcPPTWPRE,采用磷酸钙法分别将pcPPTWPRE和pcPPTPRE(+)转染HEK293细胞和DF-1细胞,利用荧光显微镜观察EGFP蛋白的表达,利用流式细胞仪检测转染细胞中EGFP阳性细胞的表达率.[结果]在HEK293细胞中,pcPPTWPRE表达外源基因的能力极显著优于pcPPTPRE(+);在DF-1细胞中,pcPPTWPRE与pcPPTPRE(+)表达外源基因的能力均不强,但前者略优于后者.[结论]土拨鼠肝炎病毒转录后,调控元件(WPRE)可以明显增强EIAV载体质粒的外源蛋白表达能力,为高表达能力伪型EIAV重组病毒的获得奠定了基础,同时也为其他病毒载体的构建提供了借鉴. |
| 关键词: 马传染性贫血病毒；土拨鼠肝炎病毒 转录后调控元件；基因转移载体；增强绿色荧光蛋白 |
| DOI： |
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| 基金项目:国家科技基础条件平台工作重点项目（2004DKA30670） |
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| Optimization of the posttranscriptional regulatory element of gene transfer vector derived from Equine Infectious Anemia Virus |
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| Abstract: |
| 【Objective】 In order to improve the EGFP expression level of EIAV gene transfer vector pcPPTPRE(+),its posttranscriptional regulatory element was optimized.【Method】 The woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) was amplified by polymerase chain reaction（PCR）from the plasmid containing WPRE.The PCR product was subcloned into pGM-T to construct recombinant plasmid pGM-WPRE and sequenced.Then the WPRE was subcloned from pGM-WPRE into pcPPTPRE(+) using NotⅠto construct recombinant plasmid pcPPTWPRE.Finally the EIAV gene transfer vectors pcPPTPRE(+) and pcPPTWPRE were transfected into HEK293 cells and DF-1 cells using Calcium and Phosphate Transfection Kit respectively.The cells were investigated under fluorescence microscope at 12,24,36 and 48 h after transfection.Flow-cytometric analysis was carried out after 48 h.【Result】 Results showed that the pcPPTWPRE could express the EGFP much more efficiently than pcPPTPRE(+) in HEK293 cells,but the pcPPTWPRE could express the EGFP a little efficiently than pcPPTPRE(+) in DF-1 cells.【Conclusion】 The WPRE could enhance the EGFP expression level of EIAV vector.It was successful to improve the PRE of pcPPTPRE(+) and to obtain a more efficient EIAV vector pcPPTWPRE. |
| Key words: equine infectious anemia virus (EIAV) woodchuck hepatitis virus posttranscriptional regulatory element gene transfer vector EGFP |
