Wetzler L M, Gotschlich E C, Blake M S, Koomey J M
Rockefeller University, New York 10021.
J Exp Med. 1989 Jun 1;169(6):2199-209. doi: 10.1084/jem.169.6.2199.
Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin-resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination.
蛋白质III（PIII）是一种高度保守、抗原性稳定的淋球菌外膜蛋白，与主要外膜蛋白蛋白质I（PI）密切相关。我们之前报道过PIII基因的克隆。该基因被插入失控质粒pMOB45的Eco RI位点。来自淋球菌质粒pFA3的β-内酰胺酶（βla）Bam HI限制片段被插入PIII基因的Xba I位点。质粒构建体用Hae III进行甲基化，PIII/βla插入片段用Eco RI切下并用于转化淋球菌菌株F62。分离出一个βla+、氨苄青霉素抗性转化体并命名为2D。用亲和纯化的多克隆PIII抗血清对2D全细胞裂解物进行蛋白质印迹分析。未检测到PIII反应性。对用Eco RI或Cla I切割的F62和2D染色体DNA进行Southern印迹分析。一个PIII DNA探针与2D菌株中比F62菌株大2.2 kb的片段杂交。这与βla插入片段的大小相对应。一个βla特异性探针与上述相同的2D限制片段杂交，但不与任何F62片段反应，证实发生了同源重组。2D与其亲本菌株F62之间的表型变化极小。2D的染色体DNA能够转化淋球菌菌株F62、UU1和Pgh 3-2，使这些菌株变为PIII-。2D和其他PIII-转化体现在可用于研究PIII在淋球菌生理学、代谢、膜结构和发病机制中的作用。此外，我们现在有了可以纯化无PIII污染的淋球菌蛋白的生物体。
