Cloning, expression and purification of dust mite allergen Der f24 and identification its immunological characteristics
QIU Cong-ling1, ZHONG Xiao-jun1, 2, Huang Yi3, LIU Xiao-yu1, 2, CAI Ze-lang1, 2, LIU Zhi-gang1, 2, JI Kun-mei1, 2
1.Institute of Allergy and Immunology, School of Medicine, Shenzhen University, Shenzhen 518060, China;#br#
2.Shenzhen Key Laboratory of Allergy and Immunology, Shenzhen 518060, China;#br#
3.The first affiliated hospital of shenzhen university ,Shenzhen 518060, China
Abstract:To clone and express cDNA of dust mite(Dermatophagoides farinae,Der f) allergen Der f24, and study its immunological characteristics. Referring to the sequence data in GenBank (Accession No.KC669700), the specific primers were designed and the cDNA of Der f24 was amplified from the cDNA library. The prokaryotic expression vector pET-His-Derf24 was constructed and expressed by the induction of IPTG in E.coli BL21(DE3)plysS.The inclusion bodies were purified successively by way of denaturation,renaturation and Ni2+-Chelating chromatography. IgE-Western blotting was implemented to characterize allergenicity of the recombinant Der f24.The 3D structure of Der f24 was constructed by the Swiss-Model software. The potential B cell epitopes of Der f24 were predicted by DNASTAR V7.1. Meanwhile,three epitope peptides were synthesised and subjected to IgE-ELISA. The full length cDNA of Der f24 was cloned with 357 bp and encoding 118 amino acids(GenBank accession No. KP064571). Der f24 was highly expressed as inclusion bodies in E.coli. Recombinant Der f24 showed IgE-binding on immunoblot with the serum of all 10(100%) Der f-allergic patients tested,but was not bound by IgE antibodies in the serum obtained from any of 10 non-allergic healthy controls. IgE-ELISA demonstrated IgE binding to each of the three peptide epitopes of Der f24 (No.1 :75-88 aa,Z=5.6734;No.2: 44-57 aa,Z=5.6720;No.3 :104-117 aa,Z=5.6791;all P<0.0001) in 22 (100%) out of 22 samples. The cDNA of Der f24 from Dermatophagoides farinae mite was cloned and expressed in E.coli .Recombinant Der f24 and its three epitopes showed IgE binding activities.
邱聪龄, 钟小军, 黄毅, 刘晓宇, 蔡泽浪, 刘志刚, 吉坤美. 粉尘螨第24组过敏原cDNA克隆表达及其免疫学特性的研究[J]. 中国人兽共患病学报, 2015, 31(5): 453-457.
QIU Cong-ling, ZHONG Xiao-jun, Huang Yi, LIU Xiao-yu, CAI Ze-lang, LIU Zhi-gang, JI Kun-mei. Cloning, expression and purification of dust mite allergen Der f24 and identification its immunological characteristics. Chinese Journal of Zoonoses, 2015, 31(5): 453-457.
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