Mhashilkar A M, Bagley J, Chen S Y, Szilvay A M, Helland D G, Marasco W A
Department of Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
EMBO J. 1995 Apr 3;14(7):1542-51. doi: 10.1002/j.1460-2075.1995.tb07140.x.
Genes encoding the rearranged immunoglobulin heavy and light chain variable regions of anti-HIV-1 Tat, exon 1 or exon 2 specific monoclonal antibodies have been used to construct single chain intracellular antibodies 'intrabodies' for expression in the cytoplasm of mammalian cells. These anti-Tat single chain intrabodies (anti-Tat sFvs) are additionally modified with a C-terminal human C kappa domain to increase cytoplasmic stability and/or the C-terminal SV40 nuclear localization signal to direct the nascent intrabody to the nuclear compartment, respectively. The anti-Tat sFvs with specific binding activity against the N-terminal activation domain of Tat, block Tat-mediated transactivation of HIV-1 LTR as well as intracellular trafficking of Tat in mammalian cells. As a result, the transformed lymphocytes expressing anti-Tat sFvs are resistant to HIV-1 infection. Thus, these studies demonstrate that stably expressed single chain intrabodies and their modified forms can effectively target molecules in the cytoplasm and nuclear compartments of eukaryotic cells. Furthermore, these studies suggest that anti-Tat sFvs used either alone or in combination with other genetically based strategies may be useful for the gene therapy of HIV-1 infection and AIDS.
编码抗HIV-1反式激活因子（Tat）、外显子1或外显子2特异性单克隆抗体重排免疫球蛋白重链和轻链可变区的基因已被用于构建单链细胞内抗体（“胞内抗体”），以便在哺乳动物细胞的细胞质中表达。这些抗Tat单链胞内抗体（抗Tat sFvs）还分别用C端人κ轻链恒定区进行修饰以提高细胞质稳定性，或用C端SV40核定位信号将新生的胞内抗体导向核区室。具有针对Tat N端激活域特异性结合活性的抗Tat sFvs，可阻断Tat介导的HIV-1长末端重复序列（LTR）反式激活以及Tat在哺乳动物细胞中的细胞内运输。结果，表达抗Tat sFvs的转化淋巴细胞对HIV-1感染具有抗性。因此，这些研究表明，稳定表达的单链胞内抗体及其修饰形式可有效靶向真核细胞细胞质和核区室中的分子。此外，这些研究表明，单独使用或与其他基于基因的策略联合使用的抗Tat sFvs可能对HIV-1感染和艾滋病的基因治疗有用。
