Milan D, Nicolas J F
Unité de Biologie Moléculaire du Développement, Institut Pasteur, Paris, France.
J Virol. 1991 Apr;65(4):1938-45. doi: 10.1128/JVI.65.4.1938-1945.1991.
The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.
具有复制能力的牛白血病病毒（BLV）已被改造用作外源基因的载体。该病毒的gag、pol、env和pX区域被外源核定位信号LacZ（nlsLacZ）或SVnlsLacZ基因取代。用这种病毒DNA转染表达野生型前病毒所有BLV蛋白的绵羊细胞系FLK-BLV，产生的病毒滴度为10(4) CFU/ml。包含大部分gag区域并未显著提高滴度。构建了依赖激活剂和不依赖激活剂的逆转录病毒。在依赖激活剂的载体中，插入片段的表达依赖于Tax蛋白的存在，Tax蛋白可激活BLV长末端重复序列。在不依赖激活剂的载体中，由于存在内部启动子，插入片段的表达是组成型的。重组逆转录病毒感染受到特异性中和抗体的抑制。转导的遗传物质结构未发生重排。编码报告基因nlsLacZ的BLV载体，其产物可在单细胞中检测到，极大地简化了对其生物学特性的研究。对BLV载体宿主范围的测定表明，基于BLV的重组逆转录病毒在多种物种和细胞类型的基因转导中有效。
