屋尘螨8类变应原(Der p 8)克隆表达、纯化及免疫原性鉴定

Cloning,expression,purification and immunogenicity of the Der p 8 gene from Dermatophagoides pteronyssinus

目的对屋尘螨(Dermatophagoides pteronyssinus)8类变应原Der p 8进行克隆表达、纯化及免疫原性分析,并用生物信息学方法分析其同源性。方法根据已知Der p 8基因序列,设计PCR引物;提取屋尘螨总RNA,逆转录聚合酶链式反应(RT-PCR)扩增得到Der p 8的cDNA片段,以cDNA和设计的引物进行PCR扩增;扩增产物连接至T载体并转化入大肠埃希菌(E.coli Top10)中,培养后挑选阳性单克隆菌落测序。测序正确后,转入表达载体pET-32a,经酶切鉴定、测序后转入表达菌BL21,大量表达重组蛋白Der p 8。用亲和层析法纯化后进行免疫印迹分析(Western blotting)。利用生物信息学方法分析Der p 8基因的同源性。结果酶切鉴定显示Der p 8表达载体构建成功,目的基因分子量约为700 bp;SDS-PAGE电泳显示Der p8重组蛋白成功表达,分子量约为38 kD,且主要以包涵体形式存在。Western blotting结果显示Der p 8重组蛋白具有免疫原性。同源性分析结果显示本实验克隆Der p 8基因与NCBI基因库的Der p 8基因同源性为76.97%。结论本实验成功克隆表达并纯化出具有免疫原性的Der p 8重组蛋白,为临床上应用重组蛋白于诊断和治疗提供理论基础。

With the development of the social economy,people are increasingly experiencing allergic diseases.Dermatophagoides pteronyssinus,the house dust mite,is an important allergen causing many allergic diseases.The aim of this study was to clone,express,purify and analyze the immunogenicity of Der p 8 from Dermatophagoides pteronyssinus,and to analyze its genetic homology by bioinformatics.According to the known Der p 8 gene sequences,primers were designed.The total RNA of house dust mites was extracted.After amplification of cDNA fragments of Der p 8 by RT-PCR,the amplified products were connected to T carriers and transformed into E.coli Top10 cells.Properly sequenced genes were ligated into the expression vector pET-32a,digested by restriction enzymes and used to transform the expression strain BL21.After amplification,a large number of recombinant proteins was expressed.The expressed products were purified by affinity chromatography,and their immunological activity was assessed with Western blotting.Der p 8 was successfully connected to the expression carrier,and SDS-PAGE electrophoresis indicated successful expression.The purified Der p 8 recombinant protein’s molecular weight was approximately 38 kD,and Western blotting results indicated itsim munogenicity.Bioinformatic analysis revealed that the cloned Der p 8 gene had 76.97%sequence identity to the Der p 8 gene in the NCBI database.The recombinant Der p 8 protein with immunogenicity was successfully cloned,expressed and purified.It may be used in the specific diagnosis of dust mite allergic diseases or in specific immunotherapy for dust mite allergies,thus laying a foundation for the production of a recombinant allergen vaccine.