Alexenko A P, Izotova L S, Kostrov S V
All-Union Research Institute of Genetics and Selection of Industrial Microorganisms, Ministry of Medical Industry of the USSR, Moscow.
Biomed Sci. 1991;2(4):403-9.
An epitope of human leukocyte alpha interferon A (IFN-A), which is recognized by the murine monoclonal antibody NK2, has been mapped by using four successive approaches. Limited proteolysis of the IFN-A chain, followed by electrophoresis, Western blotting, and probing of the proteolytic fragments with NK2 showed that an epitope was located within the sequence residues 110-140. A panel of human IFN subtypes bearing substitutions within the sequence 110-140 was tested for reactivity with NK2 in enzyme-linked immunosorbent assays. The results from these assays suggested that the epitope is within the sequence 112-121. Analysis of a hybrid protein IFN-A(1-92)/F(93-166) revealed that the N-terminal region of IFN-A played no significant role in NK2 binding. Three residues of IFN-F (Asn113, Val114, and Lys121) were substituted for the corresponding residues from IFN-A (Lys113, Glu114, and Arg121) by site-directed mutagenesis of the gene encoding IFN-F. NK2 was able to bind the mutated protein, IFN-F(A 113, 114, 121), as well as unmodified IFN-A. The data show that the epitope recognized by NK2 is located within the C-terminal region of IFN-A (residues 112-121). This epitope consists of the essential residues 114 and 116, and residues 112, 113, 115, 117, and 121 presumably contribute the configuration of the epitope.
利用四种连续的方法，已对鼠单克隆抗体NK2所识别的人白细胞α干扰素A（IFN - A）的一个表位进行了定位。对IFN - A链进行有限的蛋白酶解，随后进行电泳、蛋白质印迹，并使用NK2对蛋白水解片段进行检测，结果表明一个表位位于序列残基110 - 140内。在酶联免疫吸附测定中，检测了一组在序列110 - 140内带有取代的人IFN亚型与NK2的反应性。这些测定的结果表明该表位在序列112 - 121内。对杂合蛋白IFN - A(1 - 92)/F(93 - 166)的分析表明，IFN - A的N端区域在NK2结合中不起重要作用。通过对编码IFN - F的基因进行定点诱变，将IFN - F的三个残基（Asn113、Val114和Lys121）替换为IFN - A的相应残基（Lys113、Glu114和Arg121）。NK2能够结合突变蛋白IFN - F(A 113, 114, 121)以及未修饰的IFN - A。数据表明，NK2所识别的表位位于IFN - A的C端区域（残基112 - 121）。该表位由必需残基114和116组成，残基112、113、115、117和121可能对表位的构象有贡献。
