Yuan Y, Lyng K, Zhang Y X, Rockey D D, Morrison R P
Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
Infect Immun. 1992 Jun;60(6):2288-96. doi: 10.1128/iai.60.6.2288-2296.1992.
Ocular and urogenital tract infections with Chlamydia trachomatis can progress to chronic inflammatory diseases that produce blindness and tubal infertility. The pathophysiology of these chronic disease conditions is thought to be immunologically mediated, and the chlamydial 60-kDa heat shock protein (hsp60) has been implicated as a major target antigen that stimulates the immunopathological response. The lack of chlamydial hsp60 antibodies and purified hsp60 has severely restricted studies to define more thoroughly the role of this protein in the immunopathogenesis of chlamydial disease. We produced a panel of antichlamydial hsp60 monoclonal antibodies (MAbs) and defined their specificities by immunoblotting against lysates of C. trachomatis, C. psittaci, and six other genera of bacteria. Three patterns of anti-hsp60 immunoreactivity were observed: chlamydial species specific, chlamydial genus specific, and cross-reactive. The epitopes recognized by these MAbs were localized within the primary amino acid sequence of hsp60 by immunoblotting against recombinant amino-terminal truncated hsp60 fusion polypeptides and then precisely mapped by use of overlapping synthetic peptides. The majority of the MAbs mapped to either the amino or the carboxyl termini of hsp60. Epitopes defining all three MAb reactivities mapped within amino-terminal residues 6 to 16. Genus-specific hsp60 MAbs mapped to epitopes located within this region and to residues 17 to 28 and 177 to 189. Antichlamydial hsp60 MAbs stained inclusions as effectively as MAbs specific for the major outer membrane protein. Homogeneous preparations of full-length recombinant chlamydial hsp60 and amino-terminal truncated recombinant hsp60 polypeptides were obtained by immunoabsorption chromatography with an hsp60 MAb reactive to the carboxyl terminus of the protein. Thus, the antichlamydial MAbs described here should be extremely useful for the specific immunodetection of hsp60 in tissues from individuals having different disease manifestations and for the purification of hsp60 or truncated hsp60 polypeptides for use in serologic and lymphocyte proliferation assays. The availability of these MAbs will facilitate studies to define more precisely the role of hsp60 in the immunopathogenesis of chlamydial disease.
沙眼衣原体引起的眼部和泌尿生殖道感染可发展为导致失明和输卵管性不孕的慢性炎症性疾病。这些慢性疾病状态的病理生理学被认为是由免疫介导的，衣原体60 kDa热休克蛋白（hsp60）被认为是刺激免疫病理反应的主要靶抗原。衣原体hsp60抗体和纯化的hsp60的缺乏严重限制了更全面地确定该蛋白在衣原体疾病免疫发病机制中作用的研究。我们制备了一组抗衣原体hsp60单克隆抗体（MAb），并通过对沙眼衣原体、鹦鹉热衣原体和其他六个细菌属的裂解物进行免疫印迹来确定它们的特异性。观察到三种抗hsp60免疫反应模式：衣原体种特异性、衣原体属特异性和交叉反应性。通过对重组氨基末端截短的hsp60融合多肽进行免疫印迹，将这些MAb识别的表位定位在hsp60的一级氨基酸序列内，然后使用重叠合成肽进行精确映射。大多数MAb映射到hsp60的氨基或羧基末端。定义所有三种MAb反应性的表位映射在氨基末端残基6至16内。属特异性hsp60 MAb映射到该区域内以及残基17至28和177至189的表位。抗衣原体hsp60 MAb对包涵体的染色效果与主要外膜蛋白特异性MAb相同。通过用对该蛋白羧基末端有反应的hsp60 MAb进行免疫吸附色谱法，获得了全长重组衣原体hsp60和氨基末端截短的重组hsp60多肽的均一制剂。因此，本文所述的抗衣原体MAb对于在具有不同疾病表现的个体组织中特异性免疫检测hsp60以及纯化用于血清学和淋巴细胞增殖试验的hsp60或截短的hsp60多肽应该极其有用。这些MAb的可用性将有助于更精确地确定hsp60在衣原体疾病免疫发病机制中的作用的研究。
