Shimizu N, Hoshino H
Department of Hygiene and Virology, Gunma University School of Medicine, Maebashi, Japan.
FEBS Lett. 1998 Jul 3;430(3):333-7. doi: 10.1016/s0014-5793(98)00687-5.
We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.
我们开发了一种针对1型人类免疫缺陷病毒（HIV-1）包膜（env）基因的高效定点诱变系统。为了构建用于诱变的模板质粒pS+B/MluI，将两个独立的选择标记，即一个独特的限制性酶切位点MluI和一个框内终止密码子，引入到已克隆于pUC118质粒的HIV-1毒株NL4-3的env基因V3结构域编码区。当使用诸如合成寡核苷酸或长度超过1.5 kbp的PCR扩增DNA片段等诱变引物成功突变pS+B/MluI质粒的env基因时，质粒变得对MluI消化具有抗性，并且通过抑制框内终止形成了有功能的env基因。通过该系统，即使在没有合适的限制性酶切位点的情况下，也能在短时间内准确构建HIV-1 env基因的各种定点突变体。我们在此报道的定点诱变系统将是一种精确且快速分析HIV-1 env基因等可变基因功能有用方法。
