Cloning and expression of alpha-enolase gene from Dermatophagoides farina and identification of its N-terminal B-cell epitope
LUO Wen-li1, QIU Cong-ling1, YU Wei-jia1, LIU Zhi-gang1, 2, JI Kun-mei1, 2, CAI Ze-lang1, 2
1. Institute of Allergy and Immunology, School of Medicine, Shenzhen University, Shenzhen 518060, China; 2. Shenzhen Key Laboratory of Allergy and Immunology, Shenzhen 518060, China
Abstract:We aimed to clone and express cDNA of alpha-enolase from the dust mite Dermatophagoides farina (Der f), and study the relationship between its N-terminal B-cell epitope and autoimmune disease. The cDNA of α-enolase was amplified from Der f cDNA library and sub-cloned into a pET-His vector to express in E. coli BL21(DE3)plysS. Recombinant α-enolase was purified and its enzyme activity was determined. The 3D structure was constructed by Swiss-Model. The N-terminal epitope peptide was synthesized and subjected to IgG-ELISA with the patients’ serum of rheumatoid arthritis vs healthy controls. Results showed that the gene encoding α-enolase was cloned from Der f (GenBank accession No. KJ421213.1). The pET-ENOA expression vector was constructed. High pure recombinant α-enolase was obtained with enzyme activity. ELISA demonstrated IgG binding to the citrullinated peptide from 11 of 64 rheumatoid arthritis (RA) patients’ serum samples, but was not bound by IgG antibodies from 26 healthy controls (P<0.05). The present study cloned and expressed α-enolase cDNA from the dust mite. The findings have revealed the dust mite α-enolase might be associated with human rheumatoid arthritis.
罗文丽, 邱聪龄, 余炜嘉, 刘志刚, 吉坤美, 蔡泽浪. 粉尘螨α-烯醇化酶基因的克隆表达、纯化及其N端B细胞表位的初步鉴定[J]. 中国人兽共患病学报, 2015, 31(7): 649-654.
LUO Wen-li, QIU Cong-ling, YU Wei-jia, LIU Zhi-gang, JI Kun-mei, CAI Ze-lang. Cloning and expression of alpha-enolase gene from Dermatophagoides farina and identification of its N-terminal B-cell epitope. Chinese Journal of Zoonoses, 2015, 31(7): 649-654.
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