Ro52抗原119-264位氨基酸片段克隆及其抗原性研究

Cloning of 119-264 Amino Acid Fragment of Ro52 Antigen and Analysis of Its Antigenicity

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 Ro52 antigen fragment from amino acid (AA) 119 to 264 that contains leucine zipper motif in forming the epitopes of Ro52 autoantigen. Methods cDNA encoding Ro52 antigen fragment from aa 119 to 264 was amplified by PCR from human heart cDNA first strand. The obtained cDNA fragment was inserted into vector pMTY4 and transformed into E. coli pop2136 for fusion protein expression. The recombinant fusion protein was then purified, and the antigenicity was determined by immunoblotting. Results A 28 000 Dalton fusion protein was obtained. It was highly expressed in host E. coli pop2136. 61.90% of the anti-full-length Ro52 positive sera reacted with the fusion protein in immunoblotting. Conclusion The results suggest that the antigen fragment from the 119 AA to 264 AA that contains the leucine zipper motif represents an important epitope region in Ro52 autoantigen.