Objective: This research aims to verify Keratin 8 (K8) as a specific autoantigen in rheumatoid arthritis (RA). Methods: First, total RNA was extracted from HaCaT cell to obtain cDNA by inverse transcription. Then, PCR was performed to amplify corresponding gene by K8 primers. Next, cloning, expression, and purification technology were used to obtain the recombinant human 1(8 (rhK8). At last, the purified rhK8, after identified by mass spectrometer, was used to perform further disease-related Western blotting and ELISA test with real clinical samples. Results: Purified rhK8 was successfully obtained and then Western blotting confirmed antigenicity of K8 in rheumatoid arthritis. The reactivity of serum IgG against rhK8 was further detected in 34 of 50 RA patients (68%). The reactivity of RA serum IgG antibodies against K8 was significantly higher than healthy controls and systemic lupus erythematosus (SLE) patients. Conclusion: This research confirmed Keratin 8 as a novel autoantigen of RA. (C) 2015 Elsevier Inc. All rights reserved.