Leonard C J, Berns K I
Hearst Microbiology Research Center, Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Virology. 1994 May 1;200(2):566-73. doi: 10.1006/viro.1994.1219.
Using the vaccinia virus/T7-RNA polymerase transient protein expression system, the AAV Rep78 protein was expressed in mammalian cells. Rep78 protein was found localized primarily to the nucleus of cells. Maximal steady-state protein levels were reached as early as 12 hr postinfection, with no discernable increase at later time points. The Rep78 protein has been partially purified from nuclear extracts of the expression system. We have successfully used the cloned, purified Rep78 protein to complement an uninfected HeLa cell extract in an in vitro AAV DNA replication assay. Rep78-containing fractions are sufficient to make an uninfected HeLa cell extract competent for AAV DNA replication.
利用痘苗病毒/T7-RNA聚合酶瞬时蛋白表达系统，AAV Rep78蛋白在哺乳动物细胞中得以表达。发现Rep78蛋白主要定位于细胞核。早在感染后12小时就达到了最大稳态蛋白水平，在随后的时间点没有明显增加。Rep78蛋白已从表达系统的核提取物中部分纯化。我们已成功使用克隆、纯化的Rep78蛋白在体外AAV DNA复制试验中补充未感染的HeLa细胞提取物。含有Rep78的组分足以使未感染的HeLa细胞提取物具备AAV DNA复制能力。
