New England BioLabs, Inc., Beverly, Massachusetts 01915, and Department of Cell Biology, National Institute for Basic Biology, Okazaki, Aichi 444, Japan.
Appl Environ Microbiol. 1990 Nov;56(11):3527-31. doi: 10.1128/aem.56.11.3527-3531.1990.
A fragment of the nifH gene was amplified from natural populations of Trichodesmium spp. and cloned into a maltose-binding protein (MBP) expression vector. The peptide product of the amplified 359-bp fragment of nifH was cleaved from the fusion protein, purified, and used to generate a specific antibody to the Fe protein of nitrogenase. The antiserum recognized the MBP-nitrogenase fusion protein and the cleaved nif peptide product but not MBP. The antibody cross-reacted with nitrogenase from natural populations of Trichodesmium spp. from the Caribbean Sea and with a cultured isolate from the Kuroshio waters (Trichodesmium sp. strain NIBB1067). The same nifH fragment was amplified, cloned, and sequenced from Trichodesmium sp. strain NIBB1067 and was found to be 98% identical at both the protein and DNA levels to nifH from the Caribbean populations. Three of the six nucleotide differences between the Trichodesmium sp. strain NIBB1067 and the Trichodesmium spp. nifH sequence had also been found in a second sequence from the natural populations, indicating either that there is more than one strain of Trichodesmium sp. in natural assemblages or that there are multiple copies of nifH in the genome. This DNA fragment, which is easily amplified with the polymerase chain reaction, may provide a good indicator of species relatedness without requiring extensive cloning or sequencing. Furthermore, the use of the polymerase chain reaction in combination with a MBP protein fusion vector provides a rapid method for production of highly specific sera, starting with a small amount of DNA.
从海水中的束毛藻自然种群中扩增出 nifH 基因片段，并将其克隆到麦芽糖结合蛋白（MBP）表达载体中。从扩增的 359bp nifH 片段中切离出肽产物，对其进行纯化，并将其用于生成氮固定酶 Fe 蛋白的特异性抗体。该抗血清可识别 MBP-氮固定酶融合蛋白和切离的 nif 肽产物，但不能识别 MBP。该抗体与来自加勒比海的束毛藻自然种群中的氮固定酶以及来自黑潮水域的培养分离株（束毛藻株 NIBB1067）发生交叉反应。从束毛藻株 NIBB1067 中扩增、克隆并测序了相同的 nifH 片段，在蛋白质和 DNA 水平上与加勒比海种群的 nifH 均有 98%的同源性。在 NIBB1067 株和 Trichodesmium spp. nifH 序列之间的 6 个核苷酸差异中的 3 个也在第二个自然种群序列中发现，这表明在自然组合中有不止一种束毛藻株，或者在基因组中有多个 nifH 拷贝。该 DNA 片段可通过聚合酶链反应轻松扩增，无需进行大量克隆或测序，即可作为物种亲缘关系的良好指标。此外，使用聚合酶链反应与 MBP 蛋白融合载体相结合，可提供一种从少量 DNA 开始快速生产高度特异性血清的方法。
