EIAV基因转移载体转录后调控元件的优化  

Optimization of the posttranscriptional regulatory element of gene transfer vector derived from Equine Infectious Anemia Virus

作　　者：李亚明[1] 韩凌霞[2] 曲连东[2] 司昌德[2] 秦海斌[1] 杨增岐[1] 于海波[2] 徐佳[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]中国农业科学院哈尔滨兽医研究所实验动物中心农业部实验动物质量监督检验测试中心,黑龙江哈尔滨150001

出　　处：《西北农林科技大学学报（自然科学版）》2008年第2期1-6,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家科技基础条件平台工作重点项目(2004DKA30670)

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。[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 Not I to 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.

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 

分 类 号：Q939.47[生物学—微生物学]