Song Y H, Ma J Y, Mårdh S, Liu T, Sjöstrand S E, Rask L, Borch K, Huang G C, Barnett P, McGregor A M
Dept. of Medical and Physiological Chemistry, Uppsala University, Sweden.
Scand J Gastroenterol. 1994 Feb;29(2):122-7. doi: 10.3109/00365529409090449.
Four cDNA fragments encoding different portions of the alpha-subunit of human H,K-adenosine triphosphatase (ATPase) were amplified by means of the polymerase chain reaction technique, ligated into the plasmid pGEX-2T, and expressed as glutathione S-transferase fusion proteins in Escherichia coli. The fragments A (residues 163-313), Ba (residues 360-797), Bb (residues 526-797), and C (residues 822-1031) together encompass 77% of the alpha-subunit and cover most of its cytosolic part. The reactivities of autoantibodies in the sera from patients with pernicious anaemia with the recombinant fusion proteins were analysed by immunoblotting. One autoantigenic epitope was found in the NH2-terminal part of the Ba fragment--that is, between residues 360 and 525. No epitope was detected in the other fragments. The Ba fragment was cleaved off from the glutathione S-transferase fusion protein by the action of thrombin and was then further purified. By means of enzyme-linked immunosorbent assay, 28 of 42 sera (67%) from patients with pernicious anaemia were positive against the purified Ba fragment. The present results provide a final proof that the human H,K-ATPase alpha-subunit is a major autoantigen in the parietal cell and that the major epitope is located between residues 360 to 525 on the cytosolic side of the secretory membrane.
采用聚合酶链反应技术扩增了编码人H，K - 腺苷三磷酸酶（ATP酶）α亚基不同部分的四个cDNA片段，将其连接到质粒pGEX - 2T中，并在大肠杆菌中作为谷胱甘肽S - 转移酶融合蛋白进行表达。片段A（第163 - 313位氨基酸残基）、Ba（第360 - 797位氨基酸残基）、Bb（第526 - 797位氨基酸残基）和C（第822 - 1031位氨基酸残基）共同涵盖了α亚基的77%，并覆盖了其大部分胞质部分。通过免疫印迹分析了恶性贫血患者血清中自身抗体与重组融合蛋白的反应性。在Ba片段的NH2末端部分，即第360至525位氨基酸残基之间发现了一个自身抗原表位。在其他片段中未检测到表位。通过凝血酶的作用将Ba片段从谷胱甘肽S - 转移酶融合蛋白上切割下来，然后进一步纯化。通过酶联免疫吸附测定，42例恶性贫血患者血清中有28例（67%）对纯化的Ba片段呈阳性反应。目前的结果最终证明，人H，K - ATP酶α亚基是壁细胞中的主要自身抗原，主要表位位于分泌膜胞质侧的第360至525位氨基酸残基之间。
