粉尘螨过敏原Der f 35的克隆表达及免疫学鉴定

深圳大学过敏反应与免疫学研究所,深圳大学医学部,广东深圳 518060

生物信息学; 免疫学; 粉尘螨; 过敏原Der f 35; 克隆表达; 过敏原性鉴定

Cloning,expression and immunological identification of Der f 35 allergen from Dermatophagoides farinae
CHEN Yang, CHEN Xianxiong, OUYANG Chunyan, ZHONG Yonghao, and LIU Xiaoyu

Institute of Allergy and Immunology, Department of Medicine, Shenzhen University, Shenzhen 518060, Guangdong Province, P.R.China

bioinformatics; immunology; Dermatophagoides farinae; allergen Der f 35; cloning and expression; allergen identification

DOI: 10.3724/SP.J.1249.2021.03301

备注

粉尘螨是诱导过敏性疾病的重要变应原,可诱发Ⅰ型变态反应,对其免疫原性的鉴定是研究过敏性鼻炎等过敏性疾病的基础.首次对粉尘螨过敏原基因Der f 35进行克隆表达、纯化及免疫原性鉴定,提取粉尘螨总核糖核酸(ribonucleic acid, RNA),根据已知基因序列(GenBank:LC175222.1)设计引物进行反转录聚合酶链式反应扩增,经BamH I和Xho I双酶切,连接构建pET-32a-Der f 35载体,并克隆至大肠杆菌TOP 10菌株,取1 mL克隆菌液进行测序.使用试剂盒提取高纯度质粒转至感受态细胞Rosetta(DE3)中进行诱导、表达纯化及免疫学鉴定,并进行同源性比对分析、进化树构建及二级结构预测.克隆和表达结果显示,Der f 35基因的片段长约436个碱基对(base pair, bp); 重组蛋白Der f 35相对分子质量约为30 ku(1 u =1 D),与预期结果相符.蛋白质印迹法结果证明,Der f 35与尘螨过敏性疾病患者的血清结合有明显的反应原性.生物信息学进化树结果显示,粉尘螨与屋尘螨、热带无爪螨、害嗜鳞螨、绵羊痒螨和免耳痒螨亲缘关系较近,二级结构预测显示Der f 35的氨基酸序列由2个α螺旋、6个β折叠、2个β转角和10个无规则卷曲片段组成.研究结果可为尘螨过敏性疾病的诊断与治疗提供理论依据.
Dermatophagoides farinae is an important allergen that induces allergic diseases and type I allergic reaction. The identification of its immunogenicity is the basis for the study of allergic rhinitis and other allergic diseases. First, primers were designed according to the Der f 35 gene sequence(GenBank: LC175222.1), and the total RNA was extracted from dust mite, use it as a template for Der f 35 reverse transcription polymerase chain reaction amplification. Then, the pET-32a vector was ligated with the Der f 35 gene and transfer it into Escherichia coli(E.coli)TOP 10 cloned bacteria, and 1 mL of the cloning bacteria liquid was aspirated for sequencing. The high purity plasmid was extracted and transferred to Rosetta(DE3)cells. Expression of the objective protein Der f 35 was induced, purified and immunologically identified. Homology comparison analysis, phylogenetic tree construction and secondary structure prediction was then performed. Results showed that the fragment of the Der f 35 gene was about 436 base pair(bp). The molecular weight of the recombinant protein Der f 35 was 30 ku(1 u =1 D), which was consistent with the expected results. The results of bioinformatics evolutionary tree showed that the dust mite was close to house dust mite, blomia tropicalis, scaly mite, sheep itchy mite and ear free mite. Secondary structure prediction indicated that Der f 35 amino acid sequence consists of two α helices, six β folds, two β turns and ten random coil fragments. Der f 35 gene was successfully cloned, and the coded protein was expressed and purified in E. coli with good reactivity. It lays a foundation for the study of allergic diseases of dust mites.
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