屋尘螨变应原Der p 33重组蛋白制备及血清IgE结合率

doi:10.11853/j.issn.1003.8280.2023.01.004

摘要/Abstract

摘要： 目的制备屋尘螨变应原第33组分（Der p 33）重组蛋白，并检测其与血清IgE结合率。方法参考GenBank（No.AUX14773）公布的Der p 33核酸序列设计引物，反转录-聚合酶链式反应（RT-PCR）扩增Der p 33基因，扩增产物经琼脂糖凝胶电泳鉴定后，连接pET-28a（+）载体，热转化至大肠埃希菌，挑取阳性重组质粒测序。将pET-28a（+）-Der p 33质粒转入大肠埃希菌Rosetta 2（DE3）plysS中，异丙基-β-D-硫代半乳糖苷（IPTG）大量诱导表达，柱层析纯化后，进行免疫印迹分析。采用生物信息学软件预测Der p 33的结构和理化特性。结果获得了Der p 33的编码基因，全长1 383 bp，编码460个氨基酸，相对分子质量为51 432.81。预测其二级结构包括α-螺旋（40.87%）、β-转角（7.17%）、延伸链（17.39%）和无规则卷曲（34.57%）。获得重组蛋白纯度>95.00%。免疫印迹检测重组Der p 33血清IgE结合率为18.75％（3/16）。结论成功克隆了Der p 33基因，重组蛋白有血清IgE结合活性，为屋尘螨变应原组分诊断试剂以及脱敏治疗生物制品的研发奠定了基础。

关键词: 屋尘螨, 克隆, 表达, 蛋白纯化, 生物信息学

Abstract: Objective To prepare the recombinant protein of the group 33 allergen of Dermatophagoides pteronyssinus (Der p 33), and to determine its serum IgE-binding rate. Methods The primers were designed according to the published sequence of Der p 33 (GenBank No. AUX14773), and the Der p 33 gene of D. pteronyssinus was amplified by RT-PCR. After being identified by agarose gel electrophoresis, the amplification product was connected to the pET-28a (+) vector and heat-transformed into Escherichia coli, and the positive recombinant plasmid was selected for sequencing. The pET-28a (+)-Der p 33 plasmid was transformed into E. coli Rosetta 2 (DE3) plysS, expressed with the induction by isopropyl-β-D-thiogalactoside (IPTG), and purified by column chromatography and was then subjected to Western blotting analysis. The structure and physicochemical properties of Der p 33 were predicted by bioinformatics software. Results The encoding gene of Der p 33 was obtained, which had a full length of 1 383 bp and a relative molecular weight of 51 432.81 and encoded 460 amino acids. The secondary structure was predicted to contain α-helix (40.87%), β-turn (7.17%), extended strand (17.39%), and random coil (34.57%). The purity of recombinant protein was >95.00%. According to the Western blotting analysis, the serum IgE-binding rate of recombinant Der p 33 was 18.75% (3/16). Conclusion The Der p 33 gene was successfully cloned, and the purified recombinant protein had serum IgE-binding activity, which lays a foundation for the development of diagnostic reagents for the allergen component of D. pteronyssinus and the bioproducts of desensitization treatment.

Key words: Dermatophagoides pteronyssinus, Cloning, Expression, Protein purification, Bioinformatics

中图分类号:

R384.4

杨李, 王楠, 滕飞翔, 马桂芳, 孙金霞. 屋尘螨变应原Der p 33重组蛋白制备及血清IgE结合率[J]. 中国媒介生物学及控制杂志, 2023, 34(1): 21-25.

Li YANG, Nan WANG, Fei-xiang TENG, Gui-fang MA, Jin-xia SUN. Preparation of the recombinant protein of Der p 33 from Dermatophagoides pteronyssinus and determination of its serum IgE-binding rate[J]. Chinese Journal of Vector Biology and Control, 2023, 34(1): 21-25.

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