重组 Jo-1 抗原的克隆、原核表达及其抗原特异性鉴定
[Cloning and prokaryotic expression of recombinant Jo-1 antigen and identification of its antigen specificity].
作者信息
Zhao Xiao-Yu, Bi Zhi-Li, Wu Yi-Hong, Xin Hai-Tao
机构信息
College of Life Science, Hebei University, Reseach Center for Bioengineering Technology of Hebei Province, Baoding 071002, China.
出版信息
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Dec;24(12):1170-3.
AIM
To obtain highly purified Jo-1 autoantigens.
METHODS
The full length of DNA sequence coding for Jo-1 (histidyl-tRNA synthetase) was obtained from human placenta by RT-PCR and then it was inserted into pTYB11 or pMAL-c to construct the expression vectors pTYB11-Jo-1 and pMAL-c-Jo-1. The recombinant plasmids were transformed into ER2566 and BL21 of E.coli, respectively.
RESULTS
The fusion Jo-1 antigens were expressed, Western blot analysis demonstrated they responded specifically to anti-Jo-1 antibody from the patients with autoimmune disease polymyositis and dermatomyositis, but did not respond to normal sera and 188 sera containing anti-RNP, Sm, Ro/La or RNP/Ro antibodies from rheumatosis patients.
CONCLUSION
The expressed protein of pMAL-c-Jo-1 is soluble, which accounts for more then 50% of total proteins of cells and can be purified by affinity chromatography. The purified proteins can be used as reagents for determining the anti Jo-1 antibody in the serum of patients.
目的
获取高度纯化的 Jo-1 自身抗原。
方法
通过 RT-PCR 从人胎盘中获得编码 Jo-1（组氨酰 -tRNA 合成酶）的全长 DNA 序列，然后将其插入 pTYB11 或 pMAL-c 中构建表达载体 pTYB11-Jo-1 和 pMAL-c-Jo-1。将重组质粒分别转化到大肠杆菌 ER2566 和 BL21 中。
结果
融合的 Jo-1 抗原得以表达，蛋白质免疫印迹分析表明它们对自身免疫性疾病多发性肌炎和皮肌炎患者的抗 Jo-1 抗体有特异性反应，但对正常血清以及来自风湿性疾病患者的 188 份含抗 RNP、Sm、Ro/La 或 RNP/Ro 抗体的血清无反应。
结论
pMAL-c-Jo-1 表达的蛋白是可溶的，占细胞总蛋白的 50%以上，可通过亲和层析进行纯化。纯化后的蛋白可作为检测患者血清中抗 Jo-1 抗体的试剂。
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