Niwa Mikio, Fukuoka Kazuhito, Fujimoto Takashi, Maruyama Ichiro N
Tsukuba Research Laboratory, Toagosei Co. Ltd., 2 Ohkubo, Tsukuba, Ibaraki 300-2611, Japan.
J Biotechnol. 2004 Oct 19;114(1-2):55-8. doi: 10.1016/j.jbiotec.2004.05.010.
Bacteriophage lambda surface display was used to isolate cDNA clones encoding autoantigens recognized by synovial fluid (SF) or sera from patients with rheumatoid arthritis (RA). We constructed cDNA libraries from human synovial sarcoma cells and synovial tissue, using the surface display vector lambdafoo. The cDNA libraries were screened by affinity selection using 40 SF and 44 sera as probes separately immobilized in microtiter wells. Phage clones isolated encode 13 different autoantigens; an unknown protein, two proteins previously unanalyzed as autoimmune antigens, three proteins previously unknown to be recognized by RA sera, and seven known RA antigens. When analyzed their sensitivity and specificity for RA by phage enzyme-linked immunosorbent assay, frequencies of sera that recognize the newly-isolated autoantigens ranged from 20.5 to 6.8% of a panel of RA sera, and 13.6-0% of other autoimmune disease sera. These results indicate that the lambda phage surface display may be powerful for the isolation of cDNA clones encoding autoantigens recognized by SF or sera from patients with not only RA but also other autoimmune diseases.
利用噬菌体λ表面展示技术分离编码类风湿性关节炎(RA)患者滑液(SF)或血清所识别的自身抗原的cDNA克隆。我们使用表面展示载体λfoo,从人滑膜肉瘤细胞和滑膜组织构建了cDNA文库。分别将40份滑液和44份血清作为探针固定在微量滴定板孔中,通过亲和选择筛选cDNA文库。分离得到的噬菌体克隆编码13种不同的自身抗原;一种未知蛋白、两种以前未作为自身免疫抗原分析的蛋白、三种以前未知被RA血清识别的蛋白以及七种已知的RA抗原。当通过噬菌体酶联免疫吸附测定分析它们对RA的敏感性和特异性时,识别新分离自身抗原的血清频率在一组RA血清中为20.5%至6.8%,在其他自身免疫性疾病血清中为13.6%至0%。这些结果表明,λ噬菌体表面展示技术对于分离不仅被RA患者而且被其他自身免疫性疾病患者的滑液或血清所识别的自身抗原的cDNA克隆可能很有效。