Karopoulos C, Rowley M J, Handley C J, Strugnell R A
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
J Autoimmun. 1995 Apr;8(2):235-48. doi: 10.1006/jaut.1995.0018.
Reactivity to the mycobacterial 65 kDa heat shock protein (HSP 65) has been implicated in the pathogenesis of adjuvant arthritis in the rat, and may be involved in the pathogenesis of rheumatoid arthritis or other autoimmune diseases in humans. Accordingly this study sought quantitative or qualitative differences in the antibody reactivity to HSP 65 between normal controls, patients with the multisystem autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) and patients with the mycobacterial infections, tuberculosis (TB) and leprosy. Levels of antibodies to recombinant HSP 65 in serum were measured by ELISA in normal subjects and in patients with RA, SLE, TB or leprosy. Antibody reactivity was examined by Western blotting using polypeptide fragments of HSP 65 derived by recombinant DNA techniques, or by digestion with trypsin or cyanogen bromide (CNBr). Reactivity to a synthetic peptide, the adjuvant arthritis T-cell epitope of HSP 65 (180-188), was tested by ELISA. High levels of antibodies to full length recombinant HSP 65 from Mycobacterium bovis were present in all the groups tested. By Western blot analysis, most reactivity with intact HSP 65 was retained in a 32 kDa tryptic fragment, judged by sequencing and size estimations to represent amino acid residues 118- approximately 388. This sequence included a major T-cell epitope for adjuvant arthritis (180-188), but these nine amino acids were not essential for B-cell reactivity since most sera also reacted with residues 188-540 which lack the T-cell epitope. Moreover, the 180-188 synthetic peptide was unreactive by ELISA, and did not inhibit reactivity with the intact recombinant HSP 65. In conclusion, most individuals had antibodies to mycobacterial HSP 65, presumably resulting from previous bacterial infections. The magnitude of the response was unrelated to the occurrence of systemic autoimmune disease, and the pattern of antibody reactivity with recombinant and proteolytic fragments of HSP 65 suggests that the major B-cell epitope is conformational and consists of discontinuous regions of the molecule.
对分枝杆菌65 kDa热休克蛋白（HSP 65）的反应性与大鼠佐剂性关节炎的发病机制有关，并且可能参与人类类风湿性关节炎或其他自身免疫性疾病的发病过程。因此，本研究旨在探寻正常对照组、多系统自身免疫性疾病患者（类风湿性关节炎（RA）和系统性红斑狼疮（SLE））以及分枝杆菌感染患者（结核病（TB）和麻风病）之间对HSP 65抗体反应性的定量或定性差异。通过酶联免疫吸附测定（ELISA）法检测正常受试者以及RA、SLE、TB或麻风病患者血清中针对重组HSP 65的抗体水平。使用通过重组DNA技术获得的HSP 65多肽片段，或经胰蛋白酶或溴化氰（CNBr）消化后的片段，通过蛋白质印迹法检测抗体反应性。通过ELISA法检测对合成肽（HSP 65的佐剂性关节炎T细胞表位（180 - 188））的反应性。所有受试组中均存在高水平的针对来自牛分枝杆菌的全长重组HSP 65的抗体。通过蛋白质印迹分析，与完整HSP 65的大部分反应性保留在一个32 kDa的胰蛋白酶消化片段中，通过测序和大小估计判断该片段代表氨基酸残基118至约388。该序列包含佐剂性关节炎的一个主要T细胞表位（180 - 188），但这九个氨基酸对于B细胞反应性并非必需，因为大多数血清也与缺乏T细胞表位的残基188 - 540发生反应。此外，180 - 188合成肽通过ELISA法检测无反应性，并且不抑制与完整重组HSP 65的反应性。总之，大多数个体具有针对分枝杆菌HSP 65的抗体，推测这是由既往细菌感染所致。反应的强度与系统性自身免疫性疾病的发生无关，并且与HSP 65重组和蛋白水解片段的抗体反应模式表明，主要的B细胞表位是构象性的，由分子的不连续区域组成。
