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目的研究Ro52抗原的第119至第264位氨基酸片段(含亮氨酸拉链基序)在抗原表位构成中的作用。方法用PCR法从人类心脏cDNA第一链中扩增编码第119至第264位氨基酸的基因片段,定向插入表达质粒pMTY4,转导入宿主菌pop2136诱导表达融合蛋白,重组融合Ro52抗原片段纯化后用免疫印迹法进行抗原性检测。结果重组Ro52抗原的第119至第264位氨基酸片段的融合蛋白相对分子质量为28000,该融合蛋白在宿主菌中高水平表达,形成包涵体。在42份抗Ro52抗体阳性血清中,26份(61.90%)可与重组融合蛋白反应。结论所克隆的含亮氨酸拉链基序的第119至第264位氨基酸片段是Ro52抗原的重要抗原表位区段。
Objective To investigate the role of amino acid residues 119 to 264 (containing leucine zipper motif) of Ro52 antigen in the construction of antigenic epitopes. Methods The gene fragment encoding the 119th to the 264th amino acids was amplified by PCR from the first strand cDNA of human heart and inserted into the expression plasmid pMTY4. The recombinant plasmid was introduced into the host strain pop2136 to induce the expression of the fusion protein. The recombinant fusion Ro52 antigen fragment was purified with Immunoblotting for antigenicity detection. Results As a result, the relative molecular mass of the fused protein of the 119 to 264 amino acid residues of the recombinant Ro52 antigen was 28000, and the fusion protein was highly expressed in the host bacteria to form inclusion bodies. Of the 42 anti-Ro52 antibody-positive sera, 26 (61.90%) reacted with the recombinant fusion protein. Conclusions The cloned fragment of amino acid 119 to 264 of the leucine-containing zipper motif is an important epitope fragment of Ro52 antigen.