利用重组反转录病毒表达马立克氏病病毒Meq基因

Expression of Marek′s Disease Virus Meq Gene by the Recombinant Retrovirus

为研究马立克氏病病毒（ＭＤＶ）Ｍｅｑ蛋白的生物学功能，应用磷酸钙与ＭＤＶＭｅｑ基因重组的反转录病毒转染载体质粒ＲＣＡＳ－Ｍｅｑ的ＤＮＡ，转染于鸡胚成纤维细胞系ＤＦ１。经间接免疫荧光技术研究表明，转染后第３天即开始有少量ＤＦ１细胞在细胞核表达Ｍｅｑ蛋白，以后阳性细胞增多，到转染后第７天阳性细胞最多，之后荧光即衰减；通过酶联免疫吸附试验检测，发现细胞培养上清中游离病毒在转染后第５天即可检出，直至第７天仍维持最高水平，第９天开始下降。用细胞培养上清感染ＤＦ１细胞单层，于感染后第２天即可检出Ｍｅｑ蛋白阳性细胞，之后阳性细胞逐渐增多，到第４～５天达高峰，之后逐渐衰减；培养上清中的游离病毒在感染后第４天即可被检出，第５～７天达最高值。研究结果表明：（１）磷酸钙法能很好地将重组质粒ＲＣＡＳ－ＭｅｑＤＮＡ转染到ＤＦ１细胞中，而对细胞的损伤很小；（２）转染后细胞释放出的重组病毒能感染ＤＦ１细胞并稳定地表达Ｍｅｑ蛋白。表明Ｍｅｑ基因在构建的重组病毒基因组中的存在及其转录表达都是稳定的。这为在体内和体外研究ＭＤＶＭｅｑ基因的生物学功能提供了一种非常有效的方法和途径；（３）Ｍｅｑ蛋白仅分布于细胞核（包括核质和核仁）。这一结果证实了?

Recombinant retrovirus transfer vector with the Meq gene of Marek′s disease virus (MDV) was transfected into the monolayers of chicken embryo fibroblast (CEF) cell line DF1 by co precipitating with calcium phosphate. Third day post transfection (PT), some of the cells began to express Meq protein in the nucleus (nucleoplasm and nucleolus) when detected by indirect immunofluorescence (IF) test. The percentage of the IF test positive cells was increasing and reached the peak at 7th day PT; Free viruses were detected in the supernatants of the transfected cells 5th day, reached the peak titre 7th day and began to drop 9th day PT when detected by ELISA technique. When the supernatants of the transfected cells infected the monolayers of DF1 cell, Meq protein positive cells occurred 2 days post infection and reached the peak at 4th day and declined at 6th day; free viruses in the supernatants occurred 4th day and reached the peak 5th day and declined at 9 days PT. The results demonstrated that the co precipitation of calcium phosphate with DNA was efficient for RCAS Meq plasmid to be transfected into the cells. The expression of Meq gene in the constructed recombinant retrovirus was stable. The expression of MDV Meq gene by recombinant retrovirus never been reported previously. The localization of Meq protein in the nucleus confirmed that Meq protein should be a nuclear and nucleolar protein, one of the transactivator localized in the nucleolus.