重组多肽酶联免疫吸附法检测抗Scl-70抗体的初步应用

Detection of anti-Scl-70 antibody by recombinant fusion peptide enzyme-linked immunosorbent assay:a preliminary study

目的制备人Scl-70抗原207～765位氨基酸片段融合蛋白作为自身抗原,探讨ELISA检测抗Scl-70抗体的敏感性和特异性。方法构建编码Scl-70抗原第207至第765位氨基酸片段的重组体,在宿主菌E.coliBL21(DE3)中表达融合蛋白,经Ni2+-NTA亲和层析纯化后,免疫印迹法鉴定抗原性,ELISA检测北京协和医院免疫科血清库中系统性硬化(SSc)及部分其他结缔组织病患者血清抗Scl-70抗体。结果重组融合蛋白在宿主菌中获得可溶性表达,免疫印迹法鉴定表明其能与标准抗Scl-70抗体阳性血清反应,而与正常血清、其他抗血清无反应。在36份SSc患者血清中,天然抗原免疫双扩散(DID)法共检出的6份阳性血清用重组多肽ELISA检测有5份呈阳性,30份经天然抗原DID法检测为阴性的血清用重组多肽ELISA检测有3份呈阳性;20份其他结缔组织病患者血清用重组多肽ELISA检测均为阴性。结论重组的207～765位氨基酸片段是Scl-70抗原的主要抗原表位区域,以重组多肽为基质ELISA检测抗Scl-70抗体的敏感性较高,为进一步研究抗体水平与临床病情变化的相关性奠定了基础。

Objective To detect anti-Scl-70 antibody with enzyme-linked immunosorbent assay （ELISA） using a recombinant fusion peptide which comprises 207 - 765 amino acid fragment of Scl-70 as antigen. Methods cDNA encoding Scl-70 antigen fragment from aa207 to aa765 from human placenta cDNA first strand was amplified with PCR. The obtained cDNA was inserted into expression vector Pet-g2b and transferred into E. coli BL21 （ DE3 ） for expression of his-tagged fusion peptide. After Ni2. -NTA affinity purification,the antigenicity was identified with Western blotting. Serum samples from 36 systemic sclerosis （SSc） patients, 20 patients with other connective tissue disorder （CTD） and 30 normal controls were retrospectively tested with ELISA. Results A soluble fusion peptide was expressed and purified. The antigenicity was confirmed with Western blotting using standard positive anti-Scl-70 antibody serum. Of the 36 serum samples from SSc patients ,5 of 6 samples showing positive reaction with natural Scl-70 antigen in double immunodifussion（DID） assay also recognized the recombinant fusion peptide with ELISA, only one serum sample which showed positive anti-Scl-70 in DID displayed a negative result in ELISA assay. On the contrary, 3 patients with negative anti-Scl-70 in DID showed positive results in ELISA assay using this recombinant peptide. All the serum samples from patients with other CTD showed negative results in ELISA assay. Conclusion The recombinant antigen fragment contains major epitope regions in natural Scl-70 antigen. Detection of anti-Scl-70 antibody with ELISA using the recombinant peptide can improve the sensitivity and has a potential role in determining its clinical association.