Emel'ianov V V, Balaeva N M
Mol Biol (Mosk). 1992 Jul-Aug;26(4):847-58.
Rickettsia prowazekii (virulent Breinl strain) random genomic DNA fragments were cloned in the lambda gt11 expression vector by using non-palindromic adaptors. Several immunoreactive clones were selected after screening 20,000 individual recombinant plaques with human convalescent serum. Some recombinants synthesized the complete 60 K protein, and others synthesized beta-galactosidase fusion polypeptides containing epitopes of 134 K protein of the R. prowazekii outer membrane. The amplified genomic library was screened with monospecific antibodies directed against abundant 31 K and 29.5 K outer membrane proteins. Several recombinant clones expressing full or part of 29.5 K polypeptide, and none expressing 31 K polypeptide were revealed. The serum of a patient convalescing from epidemic typhus did not react in western blot with recombinant 29.5 K protein.
用非回文衔接头将普氏立克次体(强毒株Breinl株)随机基因组DNA片段克隆于λgt11表达载体。用人恢复期血清筛选20000个单个重组噬菌斑后,挑选出几个免疫反应性克隆。一些重组体合成完整的60K蛋白,另一些合成含普氏立克次体外膜134K蛋白表位的β-半乳糖苷酶融合多肽。用针对丰富的31K和29.5K外膜蛋白的单特异性抗体筛选扩增的基因组文库。发现了几个表达完整或部分29.5K多肽的重组克隆,未发现表达31K多肽的克隆。一名斑疹伤寒恢复期患者的血清在蛋白质印迹中与重组29.5K蛋白无反应。