Xiaoxu Wang1, Peng Chen1, Jiawen Cui1, Chunhe Yang1, Hongwu Du2. 1. 112 Lab, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China. 2. 112 Lab, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing 100083, China. Electronic address: hwdu93@126.com.
Abstract
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 K8 (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.
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 humanK8 (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 RApatients (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.
Authors: Peter M Mihailovic; Wai Man Lio; Romana Herscovici; Kuang-Yuh Chyu; Juliana Yano; Xiaoning Zhao; Jianchang Zhou; Bo Zhou; Michael R Freeman; Wei Yang; Prediman K Shah; Bojan Cercek; Paul C Dimayuga Journal: PLoS One Date: 2019-02-27 Impact factor: 3.240