屋尘螨Derp23的克隆表达、纯化及过敏原性鉴定

Cloning,expression,purification,and identification of an allergen from the Der p23 gene of house dust mites

目的克隆屋尘螨Der p23基因,表达和纯化目的蛋白并进行过敏原性分析。方法提取屋尘螨总RNA,对Der p23基因进行RT-PCR(逆转录酶聚合酶链反应)扩增,经限制性内切酶双酶切,酶切产物与表达载体pET-32a(+)重组,转化入感受态大肠埃希菌溶液中。挑取阳性单克隆菌落进行扩大培养,提取目的蛋白并进行Ni2+柱亲和层析纯化,通过Western blot检测其过敏原性。通过BLAST、MEGA、SWISS-MODEL分析重组蛋白Der p23的同源性和三级结构。结果 Der p23基因片段大小约为282bp。大量表达后,SDS-PAGE电泳显示,纯化的重组蛋白Der p23的分子质量单位约为32ku,且以包涵体的形式存在于沉淀物中。Western blot显示该重组蛋白可与尘螨过敏患者血清发生反应,表明其具有过敏原性。BLAST和MEGA分析,Der p23与Der f23的氨基酸序列同源性为77%。Der p23蛋白二级结构主要有β折叠和转角结构,从中预测出5个抗原表位。结论克隆表达和纯化获得了具有过敏原性的Der p23蛋白,为尘螨过敏疫苗和诊断试剂的研制奠定了理论基础。

Objective To clone the Der p23 gene of house dust mites,express and purify the target protein,and analyze its allergenicity. Methods The total RNA of house dust mites was extracted,and the Der p23 gene was amplified using a reverse transcription-polymerase chain reaction(RT-PCR)and then digested with restriction endonuclease.The product was recombined with the expression vector pET-32 a(+)and transformed into competent E.coli cells in solution.The target protein was extracted and purified using Ni2 +column affinity chromatography.Its allergenicity was determined using Western blotting.Blast,mega,and SWISS-MODEL were used to analyze the homology and tertiary structure of the recombinant protein Der p23. Results The Der p23 gene fragment was about 282 bp in length.SDS-PAGE indicated that the molecular weight of the purified recombinant protein Der p23 was about 32 ku,and it was present as inclusion bodies in sediment.Western blotting indicated that the recombinant protein reacted with sera from patients with a dust mite allergy,indicating that it has allergenicity.Blast and mega analysis indicated that the amino acid sequence similarity of Der p23 and Der f23 was 77%.The secondary structure of Der p23 mainly consisted ofβ-folds and turns,and 5 epitopes were predicted. Conclusion An allergenic Der p23 protein was cloned,expressed,and purified.This result laid a theoretical foundation for the development of dust mite allergy vaccines and diagnostic reagents.