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使用携带绿色荧光蛋白(GFP)标记基因的山羊关节炎脑炎慢病毒进行体外跨物种感染。

In vitro cross-species infections using a caprine arthritis encephalitis lentivirus carrying the GFP marker gene.

作者信息

Mselli-Lakhal Laila, Guiguen François, Greenland Timothy, Mornex Jean-François, Chebloune Yahia

机构信息

UMR 754 INRA/ENVL/UCBL Retrovirus et Pathologie Comparée Université Lyon-1, Bâtiment B, 50, Avenue Tony Garnier, 69366 Lyon Cedex 07, France.

出版信息

J Virol Methods. 2007 Jul;143(1):11-5. doi: 10.1016/j.jviromet.2007.01.035. Epub 2007 Mar 26.

Abstract

A caprine arthritis encephalitis virus (CAEV), carrying the green fluorescent protein (GFP) into the tat region was recently reported [Mselli-Lakhal, L., Guiguen, F., Greenland, T., Mornex, J.F., Chebloune, Y., 2006. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. J. Virol. Meth. 136, 177-184]. This construct, called pK2EGFPH replicated to titres up to 10(5)IU/ml on infection of caprine cells, and could be concentrated to 10(6)IU/ml by ultracentrifugation. In the present study, the pK2EGFPH construct was characterized better and used in cross-species infection studies. The pK2EGFPH virus could transduce GFP protein expression both to goat synovial membrane cells and to an immortalized goat milk epithelial cell line. The pK2EGFPH infected cells were demonstrated to express both GFP protein and CAEV viral proteins, as demonstrated by radioimmunoprecipitation and multinucleated cell formation. However GFP expression could not be maintained over passages. This vector was used to investigate cross-species infectious potential of CAEV. The bovine cell lines MDBK and GBK were found to be sensitive to infection while the human cell lines Hela, A431 and THP-1 were not. The pK2EGFPH vector should prove useful in studies of CAEV tropism both in vitro and in vivo.

摘要

最近有报道称,一种将绿色荧光蛋白(GFP)导入tat区域的山羊关节炎脑炎病毒(CAEV)[Mselli-Lakhal, L., Guiguen, F., Greenland, T., Mornex, J.F., Chebloune, Y., 2006. 源自山羊关节炎-脑炎慢病毒的基因转移系统。《病毒学方法杂志》136, 177 - 184]。这种构建体称为pK2EGFPH,在感染山羊细胞时复制滴度高达10⁵IU/ml,并且可以通过超速离心浓缩至10⁶IU/ml。在本研究中,对pK2EGFPH构建体进行了更深入的表征,并用于跨物种感染研究。pK2EGFPH病毒能够将GFP蛋白表达转导至山羊滑膜细胞和永生化的山羊乳腺上皮细胞系。通过放射免疫沉淀和多核细胞形成证明,pK2EGFPH感染的细胞同时表达GFP蛋白和CAEV病毒蛋白。然而,GFP表达在传代过程中无法维持。该载体用于研究CAEV的跨物种感染潜力。发现牛细胞系MDBK和GBK对感染敏感,而人细胞系Hela、A431和THP-1则不敏感。pK2EGFPH载体在体外和体内CAEV嗜性研究中应会证明是有用的。

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