Liu T J, You B Y, Lin N T, Yang M T, Tseng Y H
Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan.
Biochem Biophys Res Commun. 1998 Jan 6;242(1):113-7. doi: 10.1006/bbrc.1997.7932.
The gene III protein (pIII) from phi Lf, a filamentous phage of Xanthomonas campestris pv.campestris, was purified by gel filtration with FPLC. The gIII coding region was amplified by PCR, which was then cloned into pUC18 and expressed in Escherichia coli. The size of both pIII, purified from phage particle and expressed in E. coli, is similar to the value deduced from the nucleotide sequence as shown by Western blot analysis. This is different from the case in Ff phages (f1, fd, and M13), in which the size of pIII observed in SDS-polyacrylamide gel electrophoresis is substantially larger than the deduced value. Upon infection of X. c. pv. vesicatoria carrying cloned phi Lf gIII with phi Xv, a filamentous phage of pv. vesicatoria, the progeny particles in supernatant were able to infect both pv. campestris carrying cloned phi Lf gIII and pv. vesicatoria, indicating that a mixture of authentic phi Xv and chimeric phage consisting of phi Xv DNA and phi Lf pIII was produced. These results suggest pIII to be the adsorption protein required for host recognition.
从野油菜黄单胞菌致病变种的丝状噬菌体phi Lf中提取的基因III蛋白（pIII），通过快速蛋白液相色谱法（FPLC）的凝胶过滤进行纯化。通过聚合酶链反应（PCR）扩增gIII编码区，随后将其克隆到pUC18中并在大肠杆菌中表达。经蛋白质免疫印迹分析表明，从噬菌体颗粒中纯化以及在大肠杆菌中表达的pIII大小，与从核苷酸序列推导的值相似。这与Ff噬菌体（f1、fd和M13）的情况不同，在Ff噬菌体中，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）中观察到的pIII大小明显大于推导值。用致病变种的丝状噬菌体phi Xv感染携带克隆的phi Lf gIII的野油菜黄单胞菌致病变种，上清液中的子代颗粒能够感染携带克隆的phi Lf gIII的野油菜黄单胞菌致病变种和致病变种，这表明产生了由phi Xv DNA和phi Lf pIII组成的正宗phi Xv和嵌合噬菌体的混合物。这些结果表明pIII是宿主识别所需的吸附蛋白。
