组氨酰转移核糖核酸合成酶自身抗原的原核表达及初步临床应用  

Cloning and expression of histidyl-tRNA synthetase autoantigen gene and its clinical application

作　　者：李珊珊[1] 李永哲[1] 赵智贤[2] 佟大伟[1] 张蜀澜[1] 胡朝军[1] 杨卫平[2] 

机构地区：[1]中国医学科学院北京协和医院检验科,100730 [2]生物芯片北京国家工程研究中心博奥生物公司

出　　处：《中华检验医学杂志》2008年第2期138-142,共5页Chinese Journal of Laboratory Medicine

基　　金：国家自然科学基金资助项目（30471617,30640084）;国家高技术研究发展计划重大专项基金资助项目（2002AA222011）

摘　　要：目的通过克隆人组氨酰转移核糖核酸合成酶自身抗原Jo-1基因，构建重组表达质粒，获得具有免疫活性的纯化重组蛋白，建立间接ELISA法，并探讨其在检测多发性肌炎／皮肌炎（PM／DM）中的抗Jo-1抗体的价值。方法构建重组表达载体，在大肠杆菌DH5仪和B121（DE3）中表达；融合蛋白经Ni-NTA树脂柱进行亲和层析纯化，并通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS—PAGE）和免疫印迹（WB）进行免疫活性鉴定；应用表达蛋白建立间接ELISA法。同时用该方法检测30名正常献血者，30例SLE，30例类风湿关节炎（RA），10例原发性干燥综合征（SS），75例PM／DM患者血清中抗Jo-1抗体。结果经重组质粒测序和酶切结果证实，Jo-1目的基因已正确插入原核表达载体中，基因序列正确，符合表达框架；经SDS-PAGE检测显示，表达产物在相对分子质量55000处有一明显的蛋白表达条带；WB分析表明，重组蛋白具有人Jo-1抗原反应性；间接ELISA法检测标本血清结果显示，PM／DM组中抗Jo-1抗体的阳性率为28％，非PM／DM组（疾病对照组及正常对照组）均为阴性，其差异均有统计学意义（r=31．84，均P〈0．01）。结论成功克隆了人组氨酰转移核糖核酸合成酶自身抗原Jo-1基因，其可在大肠杆菌中表达，且重组自身抗原具有较好的抗原性和特异性。应用纯化融合蛋白建立的间接ELISA法检测PM／DM抗Jo-1抗体具有较好的特异性。Objective To clone and construct the recombinant plasmid containing Jo-1 of HepG2 cells, then purify the protein and identify the immunoreactivity of the recombinant protein, and establish the enzyme linked immunosorbent assay（ELISA）to detect Jo-1 autoantigen correlative antibodies in diagnosis of polymyositis/dermatomyositis. Methods The constructed plasmid was transformed into E. coli. DHSα and BL21 （DE3）. This fusion protein was purified by Ni-NTA chromatography and its immunoreactivity was identified by SDS-PAGE and Western blot. ELISA with the fusion protein was established to detect the Jo-1 autoantigen correlative antibodies in serum samples of 75 patient with PM/DM, 30 patients with SLE, 30 patients with RA, 10 patients with SS and 30 normal controls. Results The sequence of Jo-1 autoantigen gene was the same as the sequence reported on the literatures. SDS-PAGE gel analysis showed the molecular weight of fusion protein was approximately 55 000 Da. Western blotting analysis showed that the fusion protein had the same immunoreactivity as human Jo-1 autoantigen. The results of ELISA indicated that the positive rate of anti-Jo-1 antibody was 28%, but the antibody was negative in other controls. There was significant difference of positivity of the autoantibody between PM/DM and disease controls or normal controls （χ^2 = 31.84, P 〈 0. 01 ） . Conclusions The plasmid containing Jo-1 is successfully cloned into E. coli. DH5α and BL21 （DE3）. ELISA analysis shows its good antigenicity and specificity.

关 键 词：组氨酸tRNA连接酶 自身抗原 克隆 分子 酶联免疫吸附测定 皮肌炎 

分 类 号：R686[医药卫生—骨科学]